AU2018267631B2 - Methods of Reducing Immunogenicity Against Factor VIII in Individuals Undergoing Factor VIII Therapy - Google Patents

Abstract

The present disclosure provides methods of administering chimeric and hybrid Factor VIII (FVIII) polypeptides comprising FVIII and Fc to subjects at risk of developing inhibitory FVIII immune responses, including anti-FVIII antibodies and/or cell-mediated immunity. The administration is sufficient to promote coagulation and to induce immune tolerance to FVIII. The chimeric polypeptide can comprise full-length FVIII or a FVIII polypeptide containing a deletion, e.g., a full or partial deletion of the B domain.

Description

METHODS OF REDUCING IMMUNOGENICITY AGAINST FACTOR VIII IN INDIVIDUALS UNDERGOING FACTOR VIII THERAPY

Field of the Disclosure

[0001] The present disclosure relates generally to the field of therapeutics for hemostatic disorders.

Background Art

[0002] Hemophilia A is an X-linked bleeding disorder caused by mutations and/or deletions in the Factor VIII (FVIII) gene resulting in a deficiency of FVIII activity (Peyvandi, F. et al. Haemophilia 12:82-89 (2006). The disease is characterized by spontaneous hemorrhage and excessive bleeding after trauma. Over time, the repeated bleeding into muscles and joints, which often begins in early childhood, results in hemophilic arthropathy and irreversible joint damage. This damage is progressive and can lead to severely limited mobility of joints, muscle atrophy and chronic pain (Rodriguez-Merchan, E.C., Semin. Thromb. Hemost. 29:87-96 (2003), which is herein incorporated by reference in its entirety).

[0003] The A2 domain is necessary for the procoagulant activity of the FVIII molecule. Studies show that porcine FVIII has six-fold greater procoagulant activity than human FVIII (Lollar & Parker, J Biol. Chem. 266:12481-12486 (1991)), and that the difference in coagulant activity between human and porcine FVIII appears to be based on a difference in amino acid sequence between one or more residues in the human and porcine A2 domains (Lollar, P., et al., J Biol. Chem. 267:23652-23657 (1992)), incorporated herein by reference in its entirety.

[0004] Treatment of hemophilia A is by replacement therapy targeting restoration of FVIII activity to 1 to 5 % of normal levels to prevent spontaneous bleeding (Mannucci, P.M., et al., N. Engl. J Med. 344:1773-1779 (2001), which is herein incorporated by reference in its entirety). e.g.

[0005] Plasma-derived FVIII (pdFVIII) and recombinant human FVIII (rFVIII) products are utilized for treatment (on-demand therapy) and prevention (prophylaxis therapy) of bleeding episodes. rFVIII was developed to reduce the risk of blood-borne pathogen transmission following the widespread contamination of plasma products with HIV and hepatitis viruses, and to secure an adequate supply of FVIII product. However, hemostatic protection with current FVIII products is temporally limited due to a short half-life(t 1 /2 ) of approximately 8 12 hours, requiring prophylactic injections three times per week or every other day for most patients in order to maintain FVIII levels above 1%, a level that has been established as protective against most spontaneous bleeding episodes. Manco-Johnson et al., New Engl J Med. 357(6):535-44 (2007).

[0006] Many studies have shown that, even at high doses, on-demand therapy is not effective in preventing arthropathy. Aledort L. et al., JIntern Med. 236:391-399 (1994); Petrini P. et al., Am JPediatrHematol Oncol.13:280-287 (1991). The benefits of prophylactic therapy have been demonstrated in numerous clinical studies. Aznar J. et al., Haemophilia 6(3):170-176 (2000), Feldman B. et al., J Thromb Haemost. 4:1228-1236 (2006), Kreuz W. et al., Haemophilia4:413 417 (1998), Liesner R. et al., B J Haem. 92:973-978 (1996), Ljung R., Haemophilia. 4(4):409 412 (1998), LUfquist T, et al., JIntern Med 241:395-400 (1997), Nilsson I, et al., B. JInt Med 232:25-32 (1992), Risebrough N. et al., Haemophilia. 14:743-752 (2008), Van Den Berg H. et al., Haemophilia 9 (Suppl. 1):27-31 (2003), Van Den Berg H. et al., Haematologica89(6):645 650 (2004) and Manco-Johnson et al., supra, established that children started on primary prophylaxis after their first joint bleed had significantly fewer bleeds and less joint damage than children treated on-demand. 100071 Compared to on-demand treatment, prophylactic therapy also decreases disability, hospitalization rate, and time lost from school or work; Aznar J. et al., Haemophilia6(3):170-176 (2000), Molho P. et al., Haemophilia 6(1):23-32 (2000) and improves quality of life for patients and their families. Coppola A. et al., Blood Transfus. 6(2): 4-11 (2008). However, prophylactic therapy often requires use of central venous access devices in children, and their attendant risks of infection, sepsis, and thrombosis. In addition, despite the benefits, acceptance of and compliance with prophylaxis decreases with age, in part because of inconvenience and invasiveness. Geraghty S. et al., Haemophilia 12:75-81 (2006), Hacker M. et al., Haemophilia 7(4):392-396 (2001). Thus, an rFVIII product with a prolonged plasma t1 /2 would potentially be of benefit. Lillicrap D., CurrentOpinion in Hematology 17:393-397 (2010).

[00081 Reduced mortality, prevention of joint damage, and improved quality of life have been important achievements due to the development of pdFVIII and rFVIII. Prolonged protection from bleeding would represent another key advancement in the treatment of hemophilia A patients. However, to date, no products that allow for prolonged hemostatic protection have been developed. Therefore, there remains a need for improved methods of treating hemophilia due to FVIII deficiency that are more tolerable, longer lasting, and more effective than current therapies.

100091 In addition, 15-30% of previously untreated patients develop neutralizing anti FVIII antibodies (inhibitors) after transfusion with FVIII products. Various techniques for avoiding such immune responses have been considered. These techniques include high-dose tolerance protocols, use of peptide decoys mimicking the anti-FVIII antibody, bypassing immune recognition with human/porcine FVIII hybrid molecules, neutralizing FVIII-reactive CD4 T-cells with anticlonotypic antibodies, using universal CD4 epitopes, and blocking costimulation of CTLA-4-Ig or anti-CD40L. See, e.g., Lei et al., Transfusion Medicine 105: 4865-4870 (2005). Presentation of FVIII by immune cells in order to induce tolerance has also been studied. For example, Lei et al. found that presentation of FVIII domains on an Ig backbone in B cells prevented or decreased antibodies. Id. In addition, Qadura et al. found that tolerogenic presentation of FVIII using immature dendritic cells may reduce immunogenicity. Journal of Thrombosis and Haemostatis 6: 2095-2104 (2008). However, such methods are costly, complicated (e.g., by requiring co-administration of other therapeutics in combination with FVIII or administration of whole cells instead of relatively simple proteins), likely to result in unwanted side-effects, and/or inefficient. Accordingly, there remains a need for simple methods of treating hemophilia due to FVIII deficiency that are effective in patients that develop inhibitory responses.

BRIEF SUMMARY

[00101 The present disclosure provides methods of administering Factor VIII (FVIII) that improve immune tolerance. The methods comprise administering a chimeric polypeptide comprising a FVIII portion and an Fc portion (rFVIIIFc) to a subject at risk of developing an inhibitory FVIII immune response. In some embodiments, the subject would develop an inhibitory immune response if administered an equivalent dose of a polypeptide consisting of the FVIII portion. In some embodiments, the subject has developed an inhibitory immune response or an inhibitory FVIII immune response. The administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion can be sufficient to treat a bleeding condition and to induce immune tolerance to FVIII. The administration can be prophylactic. In some embodiments, the administration decreases the incidence of spontaneous bleeding or prevents bleeding.

[00111 The immune response can comprise inhibitory anti-FVIII antibodies. The antibody titer is at least 0.6 Bethesda Units (BU), at least 1.0 BU, or at least 5.0 BU. The immune response can also comprise a cell-mediated immune response, for example, the release of a cytokine. The cytokine can be IL-12, IL-4, or TNF-a, for example. The immune response can also result in clinical symptoms such as increased bleeding tendency, high FVIII consumption, lack of response to FVIII therapy, decreased efficacy of FVIII therapy, and/or shortened half-life of FVIII.

[00121 Subjects at risk of developing an inhibitory immune response include those with a mutation, deletion, or rearrangement in a FVIII gene. In some embodiments, the subject does not produce a FVIII protein. In some embodiments, the subject has severe hemophilia. In some embodiments, the subject has a relative (e.g., a parent, cousin, aunt, uncle, grandparent, child, or grandchild) that has developed an inhibitory immune response to FVIII or another therapeutic protein. In some embodiments, the subject is concurrently or was previously receiving a therapy that increases immune function when the FVIII is administered. In some embodiments the subject is receiving interferon therapy or anti-viral therapy in combination with FVIII. In some embodiments, the subject at risk of developing an inhibitory immune response has a genetic polymorphism associated with an increased cytokine level, such as increased TNF-aX or increased IL10. In some embodiments, the subject at risk of developing an inhibitory immune response has a TNF-a-308G>A polymorphism or allele 134 of the ILOG microsatellite.

[00131 In some embodiments, the subject at risk for developing an inhibitory FVIII immune response has not been previously exposed to FVIII. In some embodiments, the subject at risk for developing an inhibitory FVIII immune response has been exposed to FVIII. In some embodiments, the subject at risk for developing an inhibitory FVIII immune response has had less than 150, less than 50, or less than 20 FVIII exposure days. 100141 In some embodiments, the subject at risk for developing an inhibitory FVIII immune response has not previously developed an immune response to FVIII or another therapeutic protein. In some embodiments, the subject at risk for developing an inhibitory FVIII immune response has previously developed an immune response to FVIII (pdFVIII or rFVIII) or another therapeutic protein. In some embodiments, the subject at risk for developing an inhibitory FVIII immune response has previously developed an immune response to a FVIII product such as ADVATE, RECOMBINATE, KOGENATE FS*, HELIXATE FS*, XYNTHA*/REFACTO AB*, HEMOFIL-M*, MONARC-M*, MONOCLATE-P*, HUMATE-P*, ALPHANATE", KOATE-DVI, or HYATE:C*. In some embodiments, the FVIII products is a full length FVIII, a mature FVIII, or a B-domain deleted FVIII.

100151 The methods of administering FVIII provided herein can induce immune tolerance. In some embodiments, the administration reduces the number of anti-FVIII antibodies in the subject, the titer of anti-FVIII antibodies in the subject and/or the level of a cytokine (e.g., IL-12, IL-4, or TNF) in the subject compared to the number, titer, or level prior to administration. In some embodiments, the administration reduces the number of anti-FVIII antibodies in the subject, the titer of anti-FVIII antibodies in the subject and/or the level of a cytokine (e.g., IL-12, IL-4, or a TNF) in the subject compared to the number, titer, or level that resulted from a previous treatment with a polypeptide consisting of a FVIII polypeptide. In some embodiments, the administration reduces the number of anti-FVIII antibodies in the subject, the titer of anti FVIII antibodies in the subject and/or the level of a cytokine (e.g., IL-12, IL-4, or TNF) in the subject compared to the number, titer, or level that would result from administration of polypeptide consisting of a FVIII polypeptide to the subject.

[00161 The methods comprise administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion. The FVIII portion can be human FVIII, full-length FVIII, or FVIII containing a full or partial deletion of the B domain. The FVIII portion can be a biologically active polypeptide, e.g., a FVIII polypeptide with coagulation activity. The FVIII portion can be at least 90% identical, 95% identical, or identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6). The FVIII portion can also be at least 90% identical, 95% identical, or identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6). The Fc portion can be identical to the Fc amino acid sequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6).

[00171 The chimeric polypeptide can be in a form of a hybrid comprising a second polypeptide in association with the chimeric polypeptide. The second polypeptide can consist essentially of or consist of an Fc.

[00181 In some embodiments, the methods comprise administration of a chimeric polypeptide at a particular dose. The dose can be, for example, a dose of 10-100 IU/kg, a dose of 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg, or a dose of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg.

[00191 In some embodiments, the chimeric polypeptide is administered to a subject with a bleeding condition. The bleeding condition can be, for example, a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. In some embodiments, the bleeding coagulation disorder is hemophilia A.

[00201 The present disclosure also provides a method of administering a clotting factor to a subject at risk of developing an inhibitory immune response to the clotting factor comprising administering to the subject a chimeric polypeptide comprising a clotting factor portion and an Fc portion. Also provided is a method of inducing immune tolerance to a clotting factor in a subject, wherein the subject is a fetus, the method comprising administering to the mother of the fetus a polypeptide comprising a chimeric polypeptide comprising a clotting factor portion and an Fc portion. In some embodiments, the clotting factor is FVII zymogen, activated FVII, activatable FVII, or FIX.

[00211 In some embodiments, the administration of the chimeric polypeptide decreases the incidence of spontaneous bleeding or prevents bleeding. 100221 The present disclosure also provides a method of inducing immune tolerance to FVIII in a subject in need thereof, comprising administering to the subject a chimeric polypeptide comprising a FVIII portion and an Fc portion. In some embodiments, the subject is at risk of developing an inhibitory FVIII immune response. In other embodiments, the subject has developed an inhibitory Factor VIII immune response.

[00231 Also provided is method of preventing or inhibiting development of an inhibitor to FVIII, the method comprising administering to a subject in need of immune tolerance a chimeric polypeptide comprising FVIII portion and an Fc portion.

[00241 The present disclosure also provides a method of inducing immune tolerance to a clotting factor in a subject in need thereof, comprising administering to the subject a chimeric polypeptide comprising a clotting factor portion and an Fc portion. In some embodiments, the subject is at risk of developing an inhibitory clotting factor immune response. In other embodiments, the subject has developed an inhibitory clotting factor immune response. In some embodiments, the clotting factor portion comprises Factor VII, Factor IX or Von Willebrand factor.

[00251 Also provided is method of preventing or inhibiting development of an inhibitor to a clotting factor comprising administering to a subject in need thereof a chimeric polypeptide comprising a clotting factor portion and an Fc portion. In some embodiments, the clotting factor portion is Factor VII, Factor IX or Von Willebrand factor.

[00261 In some embodiments, the subject is a human and the method of administering FVIII is a method for treating a bleeding condition in said subject. In some embodiments, the bleeding condition is caused by a blood coagulation disorder. In some embodiments, the blood coagulation disorder is hemophilia or von Willebrand disease. In some embodiments, the blood coagulation disorder is hemophilia A. In some embodiments, the subject has a condition requiring prophylactic or on-demand treatment, such as a bleeding episode. In some embodiments, the subject is a patient who is suffering from a bleeding disorder or is expected to be in need of such treatment.

[00271 The present disclosure also provides a kit comprising: (a) a pharmaceutical composition comprising a chimeric polypeptide which comprises a clotting factor portion and an Fc portion or an FcRn binding partner portion and a pharmaceutically acceptable carrier, and (b) instructions to administer to the composition to a subject in need of immune toleranceto the clotting factor. In some embodiments, the chimeric polypeptide comprises a FVIII portion, a FVII portion, or a FIX portion. In some embodiments, the chimeric polypeptide is a FVIII monomer dimer hybrid, a FVII monomer dimer hybrid, or a FIX monomer dimer hybrid. In some embodiments, the instructions further include at least one step to identify a subject in need of immune tolerance to the clotting factor. In some embodiments, the step to identify the subjects in need of immune tolerance includes one or more from the group consisting of: (a) identifying a subject having a mutation or deletion in the clotting factor gene; (b) identifying a subject having a rearrangement in the clotting factor gene; (c) identifying a subject having a relative who has previously developed an inhibitory immune response against the clotting factor; (d) identifying a subject receiving interferon therapy; (e) identifying a subject receiving anti-viral therapy; (f) identifying a subject having a genetic mutation in a gene other than the gene encoding the clotting factor which is linked with an increased risk of developing an inhibitory immune response; and (g) two or more combinations thereof. In some embodiments, the genetic mutation in a gene other than the gene encoding the clotting factor comprises one or more mutations selected from the group consisting of: (a) a genetic polymorphism associated with increased TNF-a; (b) a genetic polymorphism associated with increased IL10; (c) a genetic polymorphism associated with decreased CTLA-4; (d) a mutation in DR15 or DQB0602 MHC Class II molecules; and (e) has two or more combinations thereof.

[0028] In some embodiments, the methods of the present disclosure further comprise measuring the level of an inhibitory immune response after the administration. In some embodiments, the methods of the present disclosure further comprise comparing the level of the inhibitory immune response after the administration with the level of the inhibitory immune response before the administration. In some embodiments, the inhibitory immune response is the development of antibodies against FVIII. In some embodiments, the inhibitory immune response is cytokine secretion.

[0028a] The term 'comprise' and variants of the term such as 'comprises' or 'comprising' are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

[0028b] Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0029] FIG. 1 shows a schematic representation of the rFVIIIFc monomer.

[0030] FIGS. 2A and 2B show non-reducing and reducing SDS-PAGE analysis of rFVIIIFc (processed or single chain). FIG. 2C shows the rFVIIIFc structure analyzed by LC/UV and LC/MS.

[0031] FIGS. 3A, 3B and 3C show the biochemical characterization of rFVIII-Fc. FIG. 3A shows the activation of Factor X (FX) as a function of phospholipid vesicle concentration. FIG. 3B shows the activation of FX as a function of FX concentration. FIG. 3C shows the activation of FX as a function of activated FIX (FIXa) concentration.

[0032] FIG 4 shows the activation of FX following cleavage by activated Protein C.

[0033] FIGS. 5A, 5B, 5C and 5D show observed group mean FVIII activity (±SE) versus time profiles. Profiles are sorted by dose level, grouped by compound versus time. FIG. 5A corresponds to a one stage assay with a 25 IU/kg dose. FIG. 5B corresponds to a one stage assay with a 65 IU/kg dose. FIG. 5C corresponds to a chromogenic assay with a 25 IU/kg dose. FIG. 5D corresponds to a chromogenic assay with a 65 IU/kg dose.

[0034] FIGS. 6A and 6B show observed group mean FVIII activity (±SE) versus time profiles, grouped by dose level and compound versus time. FIG. 6A corresponds to a one stage assay. FIG. 6B corresponds to a chromogenic assay.

[0035] FIGS. 7A, 7B and 7C show in vivo efficacy of single chain FVIIIFc (SC rFVIIIFc) in haemophilia A (HemA) mouse tail vein transection model. FIG. 7A shows the relationship between FVIII dose and protection. Single chain rFVIIIFc doses are shown as squares, and processed rFVIIIFc doses are shown as circles. FIG. 7B shows the percentage of survival following tail vein transection after administration of 4.6 pg/kg, 1.38 tg/kg, and 0.46 pg/kg of rFVIIIFc or SC rFVIIIFc. FIG. 7C shows the percentage of non-bleeders following tail vein

[Text continues on page 9]

- 8a - transection, after administration of 4.6 pg/kg (black circle or inverted triangle), 1.38 pg/kg (triangle or diamond), and 0.46 pg/kg (square and gray circle) of rFVIIIFc or SC rFVIIIFc, respectively.

[0036] FIG. 8 depicts the study design of the phase 1/2a study, which was a dose-escalation, sequential design to evaluate the safety and PK of rFVIIIFc compared with ADVATE* after a single intravenous dose of either 25 IU/kg (low dose Cohort A) or 65 IU/kg (high dose Cohort B). 100371 FIG. 9 shows the correlation of rFVIII Activity by one-stage (aPTT) and chromogenic assays. Results measure FVIII activity (IU/mL) following injection of ADVATE* (+) and rFVIIIFc (o).

[00381 FIGS. 1OA andlOB show group mean plasma FVIII activity pharmacokinetic profiles for low-dose and high-dose cohorts. The plasma FVIII activities (one stage aPTT assay) versus time curve after a single intravenous injection of rFVIIIFc or ADVATE* are shown for 25 IU/kg (low-dose cohort, n=6) (FIG. 1OA); and 65 IU/kg (high dose cohort, n=10 [ADVATE*]; n=9

[rFVIIIFc]) (FIG. lOB). Results presented are group mean standard error of mean (SEM). 100391 FIGS. 11A and 11 B show the effect of VWF antigen levels on Cl and 1t /2 of FVIII activity after Injection of ADVATE* or rFVIIIFc. The correlation between VWF antigen levels and the weight-adjusted Cl of ADVATE* (R2=0.5415 and p=0.0012) and rFVIIIFc (R2=0.5492 and p=0.0016) (FIG. 11A); and the t1/2 of ADVATE* (R2=0.7923 and p<0.0001) and rFVIIIFc (R2= 0.6403 and p=0.0003) (FIG. 1B) are shown. Each dot represents an individual subject.

[00401 FIGS. 12A and 12 B show ex vivo whole blood ROTEM* results for individual subjects after injection of ADVATE* or rFVIIIFc. Blood was sampled from subjects prior to and after treatment at doses of 25 IU/kg ADVATE* and rFVIIIFc (FIG. 12A); and 65 IU/kg ADVATE* and rFVIIIFc at specified time points (FIG. 12B). Clotting time was determined by NATEM initiated with Ca" on a ROTEM* instrument. Results presented are mean standard error of mean (SEM) from triplicate channel readings for each individual sample.

[00411 FIGS. 13A and 13B compare the activity of rFVIIIFc and SC rFVIIIFc in a thrombin generation assay (TGA). FIG. 13A compared endogenous thrombin potential (ETP) for rFVIIIFc, SC rFVIIIFc, fully processed rFVIIIFc and WHO standard FVIII. FIG. 13B compares peak thrombin for rFVIIIFc, SC rFVIIIFc, fully processed rFVIIIFc andWHO standard FVIII.

100421 FIG. 14 shows a schematic representation of rFVIIIFc monomer. rFVIIIFc is a recombinant fusion of human B-domain deleted FVIII with Fc from human IgGI, with no intervening linker sequence.

[00431 FIGS. 15A, 15B, 15C and 15D show pharmacokinetic (PK) profiles comparing rFVIIIFc and rFVIII in HemA mice (FIG. 15A), C57BL/6 mice (FIG. 15B), FcRn KO mice (FIG. 15C), and human FcRn transgenic Tg32B mice (FIG. 15D) following a tail vein injection of 125 IU/kg. Results shown are Mean SD from 4 mice per treatment at each time point. The PK parameter estimates are summarized in TABLE 12. 100441 FIG. 16 compares the acute activity of rFVIIIFc and rFVIII in a HemA mice tail clip bleeding model. Male HemA mice received a tail vein injection of 24 IU/kg, 72 IU/kg, or 216 IU/kg of rFVIIIFc or rFVIII followed by a 10 mm tail clip 5 minutes post dosing. Results presented are individual and median blood loss over 30 minutes following the tail clip from 20 mice in each treatment group. P<0.05 for Vehicle vs. all other treatments, and P>0.05 for C57B1/6 mice vs. HemA mice treated with 72 or 216 IU/kg of rFVIIIFc, or 216 IU/kg of rFVIII. 100451 FIGS. 17A and 17B shows the prophylactic efficacy of rFVIIIFc relative to rFVIII in the tail vein transection (TVT) bleeding model. Male HemA mice were injured by TVT either 24 hours following vehicle, rFVIII, or rFVIIIFc treatment, or 48 hours following rFVIIIFc treatment. FIG. 17A shows survival following TVT. P<0.001 by Log-Rank test of the survival curves from animals that received 12 IU/kg rFVIIIFc vs. rFVIII 24 hours prior to TVT. FIG. 17B shows rebleed within 24 hours following TVT. P=0.002 by Log-Rank test of the non-rebleed curves from animals that received 12 IU/kg rFVIIIFc vs. rFVIII 24 hours prior to TVT. 100461 FIGS. 18A and 18B show whole blood clotting time (WBCT) of rFVIIIFc and rFVIII in hemophilia A dogs. Normal WBCT range in dogs is shown by the large dashed lines. The area above the small dashed lines (20 minutes) indicates the point at which the plasma FVIII activity is expected to be below 1% of normal. FIG. 18A showsWBCT after administration of rFVIIIFc. FIG. 18B shows WBCT after administration of rFVIII followed by administration of rFVIIIFc in a crossover study.

[00471 FIGS. 19A and 19B present pharmacokinetics (PK) data for rFVIIIFc compared to rFVIII in Hemophilia A dogs after an i.v. dose. FIG. 19A shows plasma antigen concentration measured by ELISA. FIG. 19B shows plasma FVIII activity was measured by chromogenic assay. N = 4 for rFVIIIFc and N = 2 for rFVIII.

100481 FIG. 20 shows the design of a study to evaluate FVIII Immunogenicity in HemA mice. Intravenous (i.v.) dosing with rFVIIIFc; chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA*); full-length rFVIII (ADVATE*); and vehicle control took place at day 0, day 7, day 14, day 21 and day 35. Blood samples were collected a day 14, day 21, day 28, day 35 and day 42.

[00491 FIGS. 21A, 21B and 21C show anti-FVIII total antibody counts in immunogenicity experiments conducted in HemA mice according to the design study shown in FIG. 20. FIG. 21A corresponds to total anti-FVIII antibody measurements after repeated i.v. dosing with 50 IU/kg rFVIIIFc; chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA*); or full-length rFVIII (ADVATE*). FIG. 21B corresponds to total anti-FVIII antibody measurements after repeated i.v. dosing with 100 IU/kg rFVIIIFc; chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA); full-length rFVIII (ADVATE*). FIG. 21C corresponds to total anti-FVIII antibody measurements after repeated i.v. dosing with 250 IU/kg rFVIIIFc; chimeric human FVIII-murine Fc; BDD FVIII (XYNTHA*); full-length rFVIII (ADVATE®). 100501 FIGS. 22A, 22B, 22C, and 22D shows anti-FVIII antibody measurements at different times after administration of 50 IU/kg, 100 IU/kg, and 250 IU/kg doses of rFVIIIFc; chimeric human FVIII-murine Fc; BDD-FVIII (XYNTHA*); full-length rFVIII (ADVATE*). FIG. 22A shows data corresponding to samples collected at day 14. FIG. 22B shows data corresponding to samples collected on day 21. FIG. 22C shows data corresponding to samples collected on day 28. FIG. 22D shows data corresponding to samples collected on day 42.

[00511 FIG. 23 shows the correlation between total and neutralizing antibodies to FVIII. 100521 FIG. 24 shows anti-hFc antibody development after treatment with 50 IU/kg and 250 IU/kg doses of rFVIIIFc (rFVIII with human Fc).

[00531 FIG. 25 is a diagram showing the experimental procedures for the isolation and analysis of splenocytes from HemA mice in a study to measure the splenic lymphocyte response to rFVIIIFc compared with commercially available FVIII.

[00541 FIG. 26 is a diagram showing the procedure for intracellular cytokine staining using FACS (fluorescence-activated cell sorting). The procedure uses five colors, one for the CD4 lymphocyte marker, and another four colors for the IL2, IL-4, IL-10, and TNF-a cytokines.

[00551 FIGS. 27A and 27B show representative FACS dot plot. FIG. 27A corresponds to the intracellular cytokine staining of the isotype control. FIG. 27 corresponds to the intracellular cytokine staining of double positive cell containing the CD4 and TNF-a markers.

100561 FIG. 28 shows intracellular cytokine staining above control (vehicle) for CD4 and interleukin-2 (IL-2). Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA* (Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00571 FIG. 29 shows intracellular cytokine staining above control (vehicle) for CD4 and TNF-a. Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA* (Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00581 FIG. 30 shows intracellular cytokine staining above control (vehicle) for CD4 and interleukin-4 (IL-4). Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA* (Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00591 FIG. 31 shows intracellular cytokine staining above control (vehicle) for CD4 and interleukin-10 (IL-10). Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fe (mFc), XYNTHA* (Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses. 100601 FIG. 32 shows intracellular cytokine staining above control (vehicle) for CD4 and dendritic cell marker PD-Li (CD274). Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fe (mFc),XYNTHA* (Xyn), and ADVATE*®(Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00611 FIG. 33 shows intracellular cytokine staining above control (vehicle) for CD4 and dendritic cell marker CD80. Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mFc), XYNTHA* (Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00621 FIG. 34 shows intracellular cytokine staining above control (vehicle) for CD4 and Treg marker Foxp3. Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fc (mc), XYNTHA* (Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00631 FIG. 35 shows intracellular cytokine staining above control (vehicle) for CD4 and the Th inhibitory molecule Tim3. Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFe), or mouse Fc (mFc), XYNTHA*(Xyn), and ADVATE* (Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses.

[00641 FIG. 36 shows intracellular cytokine staining above control (vehicle) for CD4 and the Th inhibitory molecule CD279 (PD-1). Percentages of double positive cells were determined from FACS dot plots from all the FVIII treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group. The samples correspond to rFVIIIFc with a human Fc (hFc), or mouse Fe (mF), XYNTHA* (Xyn), and ADVATE*®(Adv). FVIII treatments were administered at 50 IU/kg and 250 IU/kg doses. 100651 FIG. 37 is a diagram showing the design of the immunogenicity study presented in Example 13. FVIII (rFVIIIFc, XYNTHA*, ADVATE*) and vehicle control were administered to HemA mice on day 0, day 7, day 14, day 21, day 35 and day 53. Blood was drawn on day 0, day 14, day 21, day 28, and day 42. Spleens were collected on day 56. FVIII was administered in 50 IU/kg, 100 IU/kg, and 250 IU/kg doses.

[00661 FIG. 38 shows the methodology used for analysis of mouse splenocytes and T-cell response profiling in Example 13. 100671 FIG. 39 shows total anti-FVIII antibody levels in blood samples collected on day 42. rFVIIIFc, BDD-rFVIII (XYNTHA*) and fl-rFVIII (ADVATE*) were administered in 50 IU/kg, 100 IU/kg, and 250 IU/kg doses.

100681 FIGS. 40A, 40B and 40C show intracellular cytokine staining of splenic CD4+ T-cells from HemA mice treated with different doses of FVIII (rFVIIIFc, BDD-rFVIII (XYNTHA*) or fl-rFVIII (ADVATE*)). Each figure shows results for IL-2 (left panel) and for TNF-t (right panel). FIG. 4A corresponds to HemA mice (N=4) injected with 50 IU/kg doses of FVIII. FIG. 4B corresponds to HemA mice (N=10) injected with 100 IU/kg doses of FVIII. FIG. 4C corresponds to HemA mice (N=4) injected with 250 IU/kg doses of FVIII.

[00691 FIG. 41 shows intracellular cytokine staining for CD4/CD25/Foxp3 triple positive splenocytes isolated from HemA mice treated with 100 IU/kg doses of rFVIIIFc, BDD-rFVIII (XYNTHA*) or fl-rFVIII (ADVATE*). 100701 FIGS. 42A-H show real time PCR for immune tolerance related cytokines in 100 IU/kg-treated HemA mice. FIG. 42A shows results for TGF-, FIG. 42B shows results for interleukin-10, FIG. 42C shows results for the IL-12a subunit of IL-35, and FIG. 42D shows results for the EBI-3 subunit of IL-35. FIG. 42E shows results for Foxp3. FIG. 42F shows results for IL2ra/CD25. FIG. 42G shows results for CTLA4. FIG. 42H shows results for IDO-1.

[00711 FIG. 43 shows FACS analysis of cells involved in the PD-Li-PD-i pathway in 100 IU/kg treated mice. Splenocytes were stained for either surface CD11cand PD-Li (FIG. 43A), or CD4 and PD-i (FIG. 43B). Bars represent percent over vehicle (*p<0.05 vs. vehicle; +p<0.05 between treatments; T-test).

[00721 FIG. 44 is a diagram showing the design of an immunogenicity comparison study for rFVIIIFc, XYNTHA* and ADVATE* in HemA Mice. FVIII doses were administered to HemA mice on day 0, day 7, day 14, day 21, and day 35. Blood was drawn on day 0, day 14, day 21, day 28, and day 42. FVIII was administered in 50 IU/kg, 100 IU/kg, and 250 IU/kg doses.

[00731 FIG. 45 shows anti-FVIII antibody measurements at 14, 21, 28 and 42 days after administration of 50 IU/kg doses of rFVIIIFc; BDD-FVIII (XYNTHA*); full-length rFVIII (ADVATE*), as well as levels of inhibitory antibodies at day 42.

[00741 FIG. 46. shows anti-FVIII antibody measurements at 14, 21, 28 and 42 days after administration of 100 IU/kg doses of rFVIIIFc; BDD-FVIII (XYNTHA); full-length rFVIII (ADVATE*), as well as levels of inhibitory antibodies at day 42.

[00751 FIG. 47 shows anti-FVIII antibody measurements at 14, 21, 28 and 42 days after administration of 250 IU/kg doses of rFVIIIFc; BDD-FVIII (XYNTHA*); full-length rFVIII (ADVATE*), as well as levels of inhibitory antibodies at day 42.

100761 FIG. 48 shows the correlation between FVIII neutralizing antibody titers and total binding antibody levels after administration of rFVIII and rFVIIIFc.

[00771 FIG. 49 is a diagram showing the T-cell response profiling component of the immunogenicity comparison study for rFVIIIFc, XYNTHA and ADVATE* in HemA mice shown in FIG. 44. Additional FVIII doses were administered to HemA mice on day 53, and spleen were collected on day 56.

[00781 FIG. 50 (right panel) shows intracellular cytokine staining for CD4/CD25/Foxp3 triple positive splenocytes isolated from HemA mice treated with 100 IU/kg doses of rFVIIIFc, BDD-rFVIII (XYNTHA*) or fl-rFVIII (ADVATE*). FIG. 50 (left panel) is a diagram showing the mechanism of action of regulatory T-cells.

[00791 FIG. 51 is a diagram showing the design of an rFVIIIFc immune tolerization study. 50 IU/kg doses of rFVIIIFc were administered i.v.to HemA mice on day 0, day 7, day, day 21, and day 35. Blood was drawn on day 0, day 14, day 21, day 28, and day 42, followed by a 1 week rest period. Mice were then rechallenged with 250 IU/kg doses of rFVIIIFc on day 49 (day 0 of rechallenge), day 56 (day 7 of rechallenge), day 63 (day 14 of rechallenge), and day 70 (day 21 of rechallenge). Blood was collected during the rechallenge on day 63 (day 14 of rechallenge), day 70 (day 21 of rechallenge), and day 77 (day 28 of rechallenge).

[00801 FIG. 52 shows total anti-FVIII antibody measurements at day 14, 21, and 28 post rechallenge with 250 IU/kg doses of rFVIIIFc. HemA mice were pretreated with 50 IU/kg rFVIIIFc or vehicle control. The results indicate that rFVIIIFc induces immune tolerance in HemA mice. 100811 FIG. 53 is a diagram showing the recycling of IgG and rFVIIIFc by FcRn. 100821 FIG. 54 is a diagram depicting cell types and cellular architecture surrounding a liver sinusoid.

[00831 FIG. 55 is a diagram showing the design of a clearance assay in which macrophage and Kupffer are depleted with CLODROSOME* (ENCAPSOME* administered as control) prior to i.v. injection of FVIII or rFVIIIFec. Three knock-out mouse models were used: HemA, DKO, and FcRn-KO. Blood samples were collected at the specified time point (4 samples per time point). 100841 FIG. 56 shows a representative staining of HemA mouse liver section with an antibody to Iba-1, a specific macrophage marker. Panels A and A' show control ENCAPSOME treatment of HemA mice. Panels B and B' show CLODROSOME* treatment of HemA mice.

Panels A' and B' show the quantification masks highlighting the stained Kupffer cells, total tissue area, and empty areas.

[00851 FIG. 57 shows an immunohistochemical (IHC) quantitative analysis of areas positively stained with a labeled antibody to F4/80 after treatment with CLODROSOME* or ENCAPSOME*, and a fluorescence-activated cell sorter (FACS) analysis identifying circulating monocytic cells in blood cells stained with the same labeled antibody toF4/80.

[00861 FIG. 58 shows RT-PCR analysis of the expression of the macrophage marker epidermal growth factor module-containing mucin-like receptor 1 (Emrl) (F4/80) in the liver, spleen, and lung of HemA mice treated with ENCAPSOME* or CLODROSOME*.

[00871 FIG. 59 shows clearance of rFVIII and rFVIIIFc in control HemA mice and macrophage/Kupffer cell depleted HemA mice.

[00881 FIG. 60 shows clearance of rFVIII and rFVIIIFc in control DKO mice (mice lacking FVIII and VWF) and macrophage/Kupffer cell depleted DKO mice.

[00891 FIG. 61 shows clearance of rFVIII and rFVIIIFc in control FcRn-KO mice (mice lacking the FcRn recycling receptor) and macrophage/Kupffer cell depleted FcRn-KO mice 100901 FIG. 62 shows Bethesda titers of mice born out of mothers immunized on gestation day 16 with the indicated FVIII drug substance or Control (untreated). Panel A shows the experimental design, depicting the timings of treatment and dose of rFVIIIFc or XYNTHA* (BDD-FVIII) to pregnant mice and pups born out of them. Panels B and C show Bethesda titers for rFVIIIFc from pups born out of rFVIIIFc-treated , XYNTHA*-treated, or Control (untreated) mice, grouped according to treatment cohort (Panel B) or grouped by individual mothers (Panel C). 100911 FIG. 63 shows Bethesda titers of mice born out of mothers immunized on gestation day 15-17 with the indicated FVIII drug substance or control (untreated). Panel A shows the experimental design, depicting the timings of treatment and dose of rFVIIIFc or XYNTHA* (BDD-FVIII) to pregnant mice and pups born out of them. Panels B and C show Bethesda titers for rFVIIIFc from pups born out of rFVIIIFc-treated, XYNTHA*-treated, or Control (untreated) mice, grouped according to treatment cohort (Panel B) or grouped by individual mothers (Panel C). 100921 FIGS. 64A and 64B show dendritic cell surface expression of CD80 and CD274, respectively, determined by staining splenocytes from mice treated with 100 IU/kg rFVIIIFc, B domain deleted FVIII (BDD-FVIII; XYNTHA*) or full length FVIII (fl-FVIII; ADVATE*) for these two antigens along with CD11c and MHC Class II. The results show percent splenocytes± SEM (n=7-9; *p<0.05 vs. vehicle; tp<0.05 vs. rFVIIIFc; T-test). FIGS. 64C and 64D show mRNA expression levels of CD274 and IDO1, respectively, determined by real time PCR from splenocytes normalized to GAPDH levels. Bars represent relative expression levels ( 2ACt)1 SEM (n=4-9; *p<0.05 vs. vehicle; tp<0.05 vs. rFVIIIFc; T-test).

[00931 FIG. 65 shows a heat map depicting the expression profiles of all the genes in a real time PCR array among the three tested groups, i.e., splenocytes of vehicle, 50 IU/kg and 250 IU/kg rFVIIIFc treated HemA mice. cDNA from each of the samples was used to monitor the expression of individual genes using a real time PCR array consisting of genes focused on tolerance and anergy associated molecules. 100941 FIG. 66 shows an expression profile of candidate genes that were identified as being up- or down- regulated by the splenocytes of 50 IU/kg rFVIIIFc treated HemA mice and comparison with the 250 IU/kg rFVIIIFc treated group. Results illustrate the change in expression of genes above the vehicle group. The cut-off for fold change in regulation was taken as 2, i.e., fold change above 2 was considered upregulation and below 0.5 as downregulation. All the candidate genes belonging to the 50 IU/kg group shown here were significantly regulated (p<0.05 vs. vehicle as well as the 250 IU/kg group; n=8-11). 100951 FIG. 67 shows a T-cell proliferation profile comparison between HemA mice receiving two weekly injections of either 50 lU/kg or 250 IU/kg of rFVIIIFc. Bars represent decrease in MFI of CFSE relative to control in T-cells + SEM (*p<0.05, T-test, n=3-5) from the 250 IU/kg and 50 IU/kg groups.

[00961 FIG. 68A-D shows IFNy secretion profiles of T-cells from HemA mice receiving two weekly injections of either 250 IU/kg of rFVIIIFc (FIG. 68A), 50 IU/kg of rFVIIIFc (FIG. 68B), 250 IU/kg of rFVIIIFc-N297A (FIG. 68C), or 50 IU/kg rFVIIIFc-N297A (FIG. 68D). Bars represent fold above vehicle of IFNy secretion + SEM (*p<0.05, T-test; n=3-5).

DETAILED DESCRIPTION

[00971 The present disclosure provides a method of treating Hemophilia A with Factor VIII (FVIII) (processed, single chain, or a combination thereof) using a longer dosing interval and/or greater AUC than is possible with currently known FVIII products. The present disclosure also provides methods of inducing immune tolerance to FVIII. The present disclosure also provides improved FVIII chimeric polypeptides and methods of production.

100981 The methods of inducing immune tolerance and production of improved chimeric polypeptides disclosed herein are also generally applicable to one or more clotting factors, e.g., FVII and FIX. Accordingly, the present disclosures regarding FVIII chimeric polypeptides (e.g., FVIIIFc) and their uses, are equally applicable to other chimeric polypeptides comprising a clotting factor portion and an Fc portion. In some specific examples, the clotting factor portion of the chimeric polypeptide is FVII or FIX. In this respect, the present disclosure provides in general a method of inducing immune tolerance to a clotting factor in a subject in need thereof comprising administering to the subject a chimeric polypeptide comprises a clotting factor portion and an Fc portion. Also provided is a method of preventing or inhibiting development of an inhibitor to a clotting factor comprising administering to a subject in need of immune tolerance to the clotting factor a chimeric polypeptide, wherein the chimeric polypeptide comprises a clotting factor portion and an Fc portion.

[00991 Treatment of hemophilia A is by replacement therapy targeting restoration of FVIII activity to 1 to 5 % of normal levels to prevent spontaneous bleeding (Mannucci, P.M. et al., N. Engl. J. Med. 344:1773-9 (2001), herein incorporated by reference in its entirety). There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on-demand or to prevent bleeding episodes from occurring by treating prophylactically. Based on the short half-life of these products (8-12 hours) (White G.C., et al., Thromb. Haemost. 77:660-7 (1997); Morfini, M., Haemophilia 9 (suppl 1):94-99; discussion 100 (2003)), treatment regimens require frequent intravenous administration, commonly two to three times weekly for prophylaxis and one to three times daily for on-demand treatment (Manco-Johnson, M.J., et al., N. Engl. J. Med. 357:535-544 (2007)), each of which is incorporated herein by reference in its entirety. Such frequent administration is painful and inconvenient. Another major challenge associated with currently available FVIII products is the development of neutralizing anti-FVIII antibodies in patients receiving FVIII therapies. Inhibitory FVIII immune responses can comprise anti-Factor VIII antibodies and/or cell-mediated immune responses.

[00100] The present disclosure provides a method of administering FVIII to a human subject in need thereof (e.g., human patient) that is at risk of developing an inhibitory FVIII immune response. The method comprises administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion.

[00101] In some embodiments, the administration of the chimeric polypeptide reduces the number of antibodies to FVIII in the subject compared to the number prior to administration. In some embodiments, the chimeric polypeptide is administered to a subject with an inhibitory FVIII immune response, and the administration can reduce the inhibitory immune response. The inhibitory FVIII immune response can be an inhibitory antibody immune response and/or a cell mediated immune response. Administration of a chimeric polypeptide according to the methods provided herein can decrease or neutralize the inhibitory immune response. Thus, in some embodiments, anti-FVIII antibodies are decreased or eliminated in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion. The decrease can be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction. 1001021 In some embodiments, the administration of the chimeric polypeptide reduces the titer of antibodies to FVIII in the subject compared to the titer prior to administration. In some embodiments, the titer of anti-FVIII antibodies is decreased in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion. Accordingly, administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion can reduce inhibitors to less than 20, less than 10, less than 5, less than 4, less than 3, less than 2, less than 1, or less than 0.6 Bethesda Units (BU). 1001031 In some embodiments, the administration of the chimeric polypeptide reduces the level of a cytokine in the subject compared to the level prior to administration. In some embodiments, cytokine levels are decreased in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion. The cytokine can be, for example, Il 12, IL-4, and/or TNF-a. The decrease can be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction.

[00104] In some embodiments, the chimeric polypeptide is administered to a subject that previously developed an inhibitory FVIII immune response. Administration of a chimeric polypeptide to such subjects, according to the methods provided herein, can result in a decreased immune response compared to the previous response. Thus, in some embodiments, fewer anti FVIII antibodies are produced in a subject after administration of a chimeric polypeptide according to the present methods than were produced after administration of a polypeptide consisting of a FVIII polypeptide. In some embodiments, the administration of the chimeric polypeptide reduces the number of antibodies to FVIII in the subject compared to the number in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

[00105] In some embodiments, the administration of the chimeric polypeptide reduces the titer of antibodies to FVIII in the subject compared to the titer in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide. In some embodiments, the titer of anti FVIII antibodies is lower in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion than was produced after administration of a polypeptide consisting of a FVIII polypeptide. In some embodiments, the administration of the chimeric polypeptide reduces the level of a cytokine in the subject compared to the level in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide. In some embodiments, cytokine levels (e.g., IL-12, IL-4, and/or TNF-a) are lower in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion than the levels after administration of a polypeptide consisting of a FVIII polypeptide. 100106] In some embodiments, the administration of the chimeric polypeptide reduces the number of anti-clotting factor antibodies in the subject compared to the number that would result from administration of a polypeptide consisting of the clotting factor portion or a polypeptide comprising the clotting factor portion, but not comprising the Fc portion to the subject. In some embodiments, the chimeric polypeptide is administered to a subject that has not previously developed an inhibitory FVIII immune response. Administration of a chimeric polypeptide to such subjects, according to the methods provided herein, can result in a lower immune response than would result from administration of a polypeptide consisting of a FVIII polypeptide. Thus, in some embodiments, fewer anti-FVIII antibodies are produced in a subject after administration of a chimeric polypeptide according to the present methods than would be produced by administration of a polypeptide consisting of a FVIII polypeptide.

[00107] In some embodiments, the administration of the chimeric polypeptide reduces the titer of anti-clotting factor antibodies in the subject compared to the titer that would result from administration of a polypeptide consisting of the clotting factor portion or a polypeptide comprising the clotting factor portion, but not comprising the Fc portion to the subject. In some embodiments, the titer of anti-FVIII antibodies is lower in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion than would be produced by administration of a polypeptide consisting of a FVIII polypeptide. In some embodiments, the administration of the chimeric polypeptide reduces the level of a cytokine (e.g., IL-12, IL-4, and/or TNF-a) in the subject compared to the level that would result from administration of a polypeptide consisting of the clotting factor portion or a polypeptide comprising the clotting factor portion, but not comprising the Fc portion to the subject. In some embodiments, cytokine levels (e.g., IL-12, IL-4, and/or TNF-a) are lower in a subject after administration of a chimeric polypeptide comprising a FVIII portion and an Fc portion than they would be after administration of a polypeptide consisting of a FVIII polypeptide.

[00108] The methods of the present disclosure can further comprise, prior to administration of the chimeric polypeptide, identifying that the subject has one or more characteristics selected from the group consisting of: (a) has a mutation or deletion in the gene encoding the clotting factor; (b) has a rearrangement in the gene encoding the clotting factor; (c) has a relative who has previously developed an inhibitory immune response against the clotting factor; (d) is receiving interferon therapy; (e) is receiving anti-viral therapy; (f) has a genetic mutation in a gene other than the gene encoding the clotting factor which is linked with an increased risk of developing an inhibitory immune response; and, (g) has two or more combinations thereof. In some embodiments, the subject has a genetic mutation in a gene other than the gene encoding the clotting factor comprises one or more mutation selected from the group consisting of: (i) a genetic polymorphism associated with increased TNF-a; (ii) a genetic polymorphism associated with increased IL10; (iii) a genetic polymorphism associated with decreased CTLA-4; (iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and, (v) two or more combinations thereof. In some embodiments, the polymorphism is associated with increased TNF-a is 308G>A. In some embodiments, the polymorphism associated with increased IL10 is allele 134 of the IL1OG microsatellite.

[00109] The present disclosure also provides a method of administering FVIII to a human subject in need thereof (e.g., human patient), comprising administering to the subject a therapeutic dose of a chimeric FVIII polypeptide, e.g., a chimeric FVIII-Fc polypeptide, or a hybrid of such a polypeptide at a dosing interval at least about one and one-half times longer than the dosing interval required for an equivalent dose of said FVIII without the non-FVIII portion (a polypeptide consisting of said FVIII portion), e.g., without the Fc portion. The present disclosure is also directed to a method of increasing dosing interval of FVIII administration in a human subject in need thereof comprising administering the chimeric FVIII polypeptide. 100110] The dosing interval can be at least about one and one-half to six times longer, one and one-half to five times longer, one and one-half to four times longer, one and one-half to three times longer, or one and one-half to two times longer, than the dosing interval required for an equivalent dose of said FVIII without the non-FVIII portion (a polypeptide consisting of said FVIII portion), e.g., without the Fc portion. The dosing interval can be at least about one and one-half, two, two and one-half, three, three and one-half, four, four and one-half, five, five and one-half or six times longer than the dosing interval required for an equivalent dose of said FVIII without the non-FVIII portion (a polypeptide consisting of said FVIII portion), e.g., without the Fc portion. The dosing interval can be about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer.

[00111] The dosing interval can be at least about one and one-half to 5, one and one-half, 2, 3, 4, or 5 days or longer.

[00112] The present disclosure also provides a method of administering FVIII to a human subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric FVIII polypeptide, e.g., a chimeric FVIII-Fc polypeptide, or a hybrid of such a polypeptide to obtain an area under the plasma concentration versus time curve (AUC) at least about one and one-quarter times greater than the AUC obtained by an equivalent dose of said FVIII without non-FVIII portion (a polypeptide consisting of said FVIII portion), e.g., without the Fc portion. The present disclosure thus includes a method of increasing or extending AUC of FVIII activity in a human patient in need thereof comprising administering the chimeric FVIII polypeptide. 100113] The present disclosure also provides a method of administering FVIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a polypeptide comprising a FVIII and an Fc or a hybrid of such a polypeptide at a dosing interval of about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer.

[00114] The methods disclosed herein can be practiced on a subject in need of prophylactic treatment or on-demand treatment. 100115] "Administering," as used herein, means to give a pharmaceutically acceptable FVIII polypeptide disclosed herein to a subject via a pharmaceutically acceptable route. Routes of administration can be intravenous, e.g., intravenous injection and intravenous infusion. Additional routes of administration include, e.g., subcutaneous, intramuscular, oral, nasal, and pulmonary administration. Chimeric polypeptides and hybrid proteins can be administered as part of a pharmaceutical composition comprising at least one excipient.

[00116] "Area under the plasma concentration versus time curve (AUC)," as used herein, is the same as the term of art in pharmacology, and is based upon the rate and extent of absorption of FVIII following administration. AUC is determined over a specified time period, such as 12, 18, 24, 36, 48, or 72 hours, or for infinity using extrapolation based on the slope of the curve. Unless otherwise specified herein, AUC is determined for infinity. The determination of AUC can be carried out in a single subject, or in a population of subjects for which the average is calculated.

[00117] A "B domain" of FVIII, as used herein, is the same as the B domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage by thrombin, e.g., residues Ser741-Arg1648 of full length human FVIII. The other human FVIII domains are defined by the following amino acid residues: Al, residues Alal-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Ile2032; Cl, residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-Cl-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glul649-Argl689, is usually referred to as the FVIII light chain activation peptide. The locations of the boundaries for all of the domains, including the B domains, for porcine, mouse and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B domain deleted FVIII" or "BDD FVIII").

[00118] An example of a BDD FVIII is REFACTO* (recombinant BDD FVIII), which has the same sequence as the FVIII portion of the sequence in TABLE 2A(i) (amino acids 1 to 1457 or 20 to 1457 of SEQ ID NO:2). In another embodiment, the B domain deleted FVIII contains an intact intracellular processing site, which corresponds to Arginine at residue 754 of B domain deleted FVIII, which corresponds to Arginine residue 773 of SEQ ID NO: 2, or residue 1648 of full-length FVIII, which corresponds to Arginine residue 1667 of SEQ ID NO: 6. The sequence residue numbers used herein without referring to any SEQ ID Numbers correspond to the FVIII sequence without the signal peptide sequence (19 amino acids) unless otherwise indicated. For example, S743/Q1638 of full-length FVIII corresponds to S762/Q1657 of SEQ ID NO: 6 due to the 19 amino acid signal peptide sequence. 1001191 A "B domain deleted FVIII" can have the full or partial deletions disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, a B domain deleted FVIII sequence comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of U.S. Patent No. 6,316,226 (also in US 6,346,513). In some embodiments, a B domain deleted FVIII has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US 6,228,620).

100120] In some embodiments, a B domain deleted FVIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US Patent No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Patent no. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent no. 5,171,844; col. 2, lines 55-68, figure 2, and example 1 of U.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Patent No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Patent no. 7,041,635; or col. 4, lines 25-53, of U.S. Patent No. 6,458,563. In some embodiments, a B domain deleted FVIII has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain (i.e., intracellular processing site), as disclosed in WO 91/09122, which is incorporated herein by reference in its entirety.

[00121] In some embodiments, a B domain deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R.C., et al J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. A B domain deleted FVIII can also contain a deletion of amino acids 771-1666 or amino acids 868 1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety. Additional B domain deletions that are part of the instant disclosure include, e.g.,: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. 83:5939-5942 (1986)), 797 through 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver et al., DNA 6:553-564 (1987)), 741 through 1648 (Pasek (PCT application No.88/00831)), 816 through 1598 or 741 through 1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions can be made in any FVIII sequence.

[00122] In one embodiment, the B domain deleted FVIII portion in the chimeric polypeptide is processed into two chains connected (or associated) by a metal bond, the first chain comprising a heavy chain (Al-A2-partial B) and a second chain comprising a light chain (A3-C1-C2). In another embodiment, the B domain deleted FVIII portion is a single chain FVIII. The single chain FVIII can comprise an intracellular processing site, which corresponds to Arginine at residue 754 of B domain deleted FVIII (residue 773 of SEQ ID NO: 2) or at residue 1648 of full length FVIII (residue 1657 of SEQ ID NO: 6).

100123] The metal bond between the heavy chain and the light chain can be any metal known in the art. For example, the metal can be a divalent metal ion. The metals that can be used to associate the heavy chain and light chain include, but not limited to, Ca2, Mn , or Cu2+ Fatouros et al., Intern. J. Pharm. 155(1): 121-131 (1997); Wakabayashi et al., JBC. 279(13): 12677-12684 (2004).

[00124] In some embodiments, the FVIII portion in the chimeric polypeptide comprises the Al domain of FVIII. In some embodiments, the FVIII portion in the chimeric polypeptide comprises the A2 domain of FVIII. In some embodiments, the FVIII portion in the chimeric polypeptide comprises the A3 domain of FVIII. In some embodiments, the FVIII portion in the chimeric polypeptide comprises the C1 domain of FVIII. In some embodiments, the FVIII portion in the chimeric polypeptide comprises the C2 domain of FVIII.

[00125] "Chimeric polypeptide," as used herein, means a polypeptide that includes within it at least two polypeptides (or subsequences or peptides) from different sources. Chimeric polypeptides can include, e.g., two, three, four, five, six, seven, or more polypeptides from different sources, such as different genes, different cDNAs, or different animal or other species. Chimeric polypeptides can include, e.g., one or more linkers joining the different subsequences. Thus, the subsequences can be joined directly or they can be joined indirectly, via linkers, or both, within a single chimeric polypeptide. Chimeric polypeptides can include, e.g., additional peptides such as signal sequences and sequences such as 6His and FLAG that aid in protein purification or detection. In addition, chimeric polypeptides can have amino acid or peptide additions to the N- and/or C-termini. 100126] In some embodiments, the chimeric polypeptide comprises a clotting factor portion (e.g., a FVIII portion) and a non-clotting factor portion (e.g., a non-FVIII portion). Exemplary non-clotting factor portions (e.g., non-FVIII portions) include, for example, Fc. Exemplary chimeric FVIII-Fc polypeptides include, e.g., SEQ ID NOs:2 or 6 (TABLE 2), with or without their signal sequences and the chimeric Fc polypeptide of SEQ ID NO:4 (TABLE 2).

[00127] As used herein, the term "portion" when applied to a chimeric polypeptide refers to one of the components or moieties of such chimeric polypeptide (e.g., "a chimeric polypeptide comprising a FVIII portion and an Fe portion," or "the FVIII portion of the chimeric polypeptide"). In other words, the term "portion" is used to indicate the source of different components in the chimeric polypeptide, but is not used to indicate a fragment of the FVIII protein or a fragment of an Fe region.

100128] The chimeric polypeptide can comprise a sequence at least 90% or 95% identical to the FVIII and Fc amino acid sequence shown in TABLE 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO:2) or at least 90% or 95% identical to the FVIII and Fc amino acid sequence shown in TABLE 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ ID NO:2), wherein the sequence has FVIII activity. The FVIII activity can be measured by activated Partial Thromboplastin Time (aPPT) assay, chromogenic assay, or other known methods. The chimeric polypeptide can comprise a sequence identical to the FVIII and Fc amino acid sequence shown in TABLE 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO:2) or identical to the FVIII and Fc amino acid sequence shown in TABLE 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ ID NO:2).

[00129] In some embodiments, the FVIII portion comprises a FVIII A3 domain. In some embodiments, the FVIII portion comprises human FVIII. In some embodiments, the FVIII portion has a full or partial deletion of the B domain. In some embodiments, the FVIII portion is at least 90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6). In some embodiments, the FVIII portion is identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 20 to 2351 of SEQ ID NO:6). The FVIII portion is at least 90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6). In some embodiments, the FVIII portion is identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6). In some embodiments, the FVIII portion has coagulation activity.

[00130] In some embodiments, the Fc portion is identical to the Fc amino acid sequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6). In some embodiments, the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide in association with the chimeric polypeptide, wherein the second polypeptide consists essentially of or consists of the Fc portion or the FcRn binding partner. 1001311 In some embodiments, the clotting factor portion of the chimeric polypeptide comprises Factor IX. In some embodiments, the Factor IX portion of the chimeric polypeptide is at least 90%, 95%, or 100% identical to a FIX amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6). In some embodiments, the chimeric polypeptide is a monomer dimer hybrid comprising a first chain comprising a FIX portion and the Fc portion and a second chain consisting essentially of or consisting of a Fc portion.

[00132] In some embodiments, the chimeric polypeptide comprises a Factor VII portion. In some embodiments, the chimeric polypeptide is a monomer dimer hybrid comprising a first chain comprising a FVII portion and the Fc portion and a second chain consisting essentially of or consisting of a Fc portion. In some embodiments, the FVII portion is inactive FVII, activated FVII, or activatable FVII.

[00133] In some embodiments, the chimeric polypeptide as disclosed herein comprises a clotting factor other than FVIII, e.g., FVII, FVIIa, or FIX. In one example, FVII is Factor VII zymogen (inactive form of FVII), activated FVII, or activatable FVII. In another example, FIX is FIX zymogen or activated FIX. A variety of non-clotting factor portions capable of increasing the half-life of a polypeptide are known in the art. 100134] In some embodiments, a chimeric polypeptide comprising a FVIII portion has an increased half-life (tl/2) over a polypeptide consisting of the same FVIII portion without the non FVIII portion. A chimeric FVIII polypeptide with an increased t1 /2 can be referred to herein as a long-acting FVIII. Long-acting chimeric FVIII polypeptides include, e.g., FVIII fused to Fc (including, e.g., chimeric FVIII polypeptides in the form of a hybrid such as a FVIIIFc monomer dimer hybrid; see Example 1, Fig. 1, and Table 2A; and US Patent Nos. 7,404,956 and 7,348,004), and FVIII fused to albumin. 100135] "Culture," "to culture" and "culturing," as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. "Cultured cells," as used herein, means cells that are propagated in vitro.

[00136] "Factor VIII," abbreviated throughout the instant application as "FVIII," as used herein, means functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. Thus, the term FVIII includes variant polypeptides that are functional. FVIII proteins can be the human, porcine, canine, and murine FVIII proteins. As described in the Background Art section, the full length polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified versions. Examples of human FVIII sequences are shown as subsequences in SEQ ID NOs:2 or 6 (TABLE 2). FVIII polypeptides include, e.g., full length FVIII, full-length FVIII minus Met at the N-terminus, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and/or FVIII with a full or partial deletion of the B domain. FVIII variants include B domain deletions, whether partial or full deletions.

[00137] A great many functional FVIII variants are known, as is discussed above and below. In addition, hundreds of nonfunctional mutations in FVIII have been identified in hemophilia patients, and it has been determined that the effect of these mutations on FVIII function is due more to where they lie within the 3-dimensional structure of FVIII than on the nature of the substitution (Cutler et al., Hum. Mutat. 19:274-8 (2002)), incorporated herein by reference in its entirety. In addition, comparisons between FVIII from humans and other species have identified conserved residues that are likely to be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632), incorporated herein by reference in its entirety.

[00138] The human FVIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337 (1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which is incorporated herein by reference in its entirety, and the amino acid sequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199, incorporated herein by reference in its entirety, discloses a recombinant DNA method for producing FVIII in mammalian host cells and purification of human FVIII. Human FVIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells) has been reported. Human FVIII has been modified to delete part or all of the B domain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each of which is incorporated herein by reference in its entirety), and replacement of the human FVIII B domain with the human factor V B domain has been performed (U.S. Pat. No. 5,004,803, incorporated herein by reference in its entirety). The cDNA sequence encoding human FVIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2, respectively, of U.S. Application Publ. No. 2005/0100990, incorporated herein by reference in its entirety.

[00139] U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein by reference in its entirety, reports functional mutants of FVIII having reduced antigenicity and reduced immunoreactivity. U.S. Pat. No. 6,376,463, Lollar, J. S., incorporated herein by reference in its entirety, also reports mutants of FVIII having reduced immunoreactivity. U.S. Application Publ. No. 2005/0100990, Saenko et al., incorporated herein by reference in its entirety, reports functional mutations in the A2 domain of FVIII.

100140] A number of functional FVIII molecules, including B-domain deletions, are disclosed in the following patents U.S. 6,316,226 and U.S. 6,346,513, both assigned to Baxter; U.S. 7,041,635 assigned to In2Gen; U.S. 5,789,203, U.S. 6,060,447, US 5,595,886, and U.S. 6,228,620 assigned to Chiron; U.S. 5,972,885 and U.S. 6,048,720 assigned to Biovitrum, U.S. 5,543,502 and U.S. 5,610,278 assigned to Novo Nordisk; U.S. 5,171,844 assigned to Immuno Ag; U.S. 5,112,950 assigned to Transgene S.A.; U.S. 4,868,112 assigned to Genetics Institute, each of which is incorporated herein by reference in its entirety. 100141] The porcine FVIII sequence is published, (Toole, J. J., et al., Proc. Nal. Acad Sci. USA 83:5939-5942 (1986)), incorporated herein by reference in its entirety, and the complete porcine cDNA sequence obtained from PCR amplification of FVIII sequences from a pig spleen cDNA library has been reported (Healey, J. F. et al., Blood 88:4209-4214 (1996), incorporated herein by reference in its entirety). Hybrid human/porcine FVIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093, incorporated herein by reference in its entirety. More recently, the nucleotide and corresponding amino acid sequences of the Al and A2 domains of porcine FVIII and a chimeric FVIII with porcine Al and/or A2 domains substituted for the corresponding human domains were reported in WO 94/11503, incorporated herein by reference in its entirety. U.S. Pat. No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA and deduced amino acid sequences. U.S. Pat. No. 6,458,563, incorporated herein by reference in its entirety assigned to Emory discloses a B-domain deleted porcine FVIII.

[00142] The FVIII (or FVIII portion of a chimeric polypeptide) can be at least 90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; and amino acids 20 to 2351 of SEQ ID NO:6), wherein said FVIII portion has FVIII activity. The FVIII (or FVIII portion of a chimeric polypeptide) can be identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; and amino acids 20 to 2351 of SEQ ID NO:6).

[00143] The FVIII (or FVIII portion of a chimeric polypeptide) can be at least 90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351 of SEQ ID NO:6), wherein said FVIII portion has FVIII activity. The FVIII (or FVIII portion of a chimeric polypeptide) can be identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids I to 1457 of SEQ ID NO:2 and amino acids I to 2351 of SEQ ID NO:6).

100144] In some embodiments, the clotting factor is a mature form of Factor VII zymogen, activated FVII (FVIIa), or activatable FVII, or a variant thereof. Factor VII (FVII, F7; also referred to as Factor 7, coagulation factor VII, serum factor VII, serum prothrombin conversion accelerator, SPCA, proconvertin and eptacog alpha) is a serine protease that is part of the coagulation cascade. FVII includes a Gla domain, two EGF domains (EGF-1 and EGF-2), and a serine protease domain (or peptidase Si domain) that is highly conserved among all members of the peptidase Sfamily of serine proteases, such as for example with chymotrypsin. FVII occurs as a single chain zymogen, an activated zymogen-like two-chain polypeptide and a fully activated two-chain form. As used herein, a "zymogen-like" protein or polypeptide refers to a protein that has been activated by proteolytic cleavage, but still exhibits properties that are associated with a zymogen, such as, for example, low or no activity, or a conformation that resembles the conformation of the zymogen form of the protein. For example, when it is not bound to tissue factor, the two-chain activated form of FVII is a zymogen-like protein; it retains a conformation similar to the uncleaved FVII zymogen, and, thus, exhibits very low activity. Upon binding to tissue factor, the two-chain activated form of FVII undergoes conformational change and acquires its full activity as a coagulation factor. Exemplary FVII variants include those with increased specific activity, e.g., mutations that increase the activity of FVII by increasing its enzymatic activity (Kcat or Km). Such variants have been described in the art and include, e.g., mutant forms of the molecule as described for example in Persson et al. 2001. PNAS 98:13583; Petrovan and Ruf. 2001. J. Biol. Chem. 276:6616; Persson et al. 2001 J. Biol. Chem. 276:29195; Soejima et al. 2001. J. Biol. Chem. 276:17229; Soejima et al. 2002. J. Biol. Chem. 247:49027. In one embodiment, a variant form of FVII includes the mutations. Exemplary mutations include V158D-E296V-M298Q. In another embodiment, a variant form of FVII includes a replacement of amino acids 608-619 (LQQSRKVGDSPN, corresponding to the 170- loop) from the FVII mature sequence with amino acids EASYPGK from the 170-loop of trypsin. High specific activity variants of FIX are also known in the art. For example, Simioni et al. (2009 N.E. Journal of Medicine 361:1671) describe an R338L mutation. Chang et al. (1988 JBC 273:12089) and Pierri et al. (2009 Human Gene Therapy 20:479) describe an R338A mutation. Other mutations are known in the art and include those described, e.g., in Zogg and Brandstetter. 2009 Structure 17:1669; Sichler etl al. 2003. J. Biol. Chem. 278:4121; and Sturzebecher et al. 1997. FEBS Lett 412:295. The contents of these references are incorporated herein by reference. Full activation, which occurs upon conformational change from a zymogen-like form, occurs upon binding to is co-factor tissue factor. Also, mutations can be introduced that result in the conformation change in the absence of tissue factor. Hence, reference to FVIIa includes a zymogen-like form, a fully activated two-chain form, or an activatable form. An "activatable Factor VII" is Factor VII in an inactive form (e.g., in its zymogen form) that is capable of being converted to an active form.

[00145] In some embodiments, the clotting factor is a mature form of Factor IX or a variant thereof. Factor IX circulates as a 415 amino acid, single chain plasma zymogen (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993)). The zymogen of FIX is activated by FXIa or by the tissue factor/FVIa complex. Specific cleavages between arginine-alanine 145-146 and arginine-valine 180-181 result in a light chain and a heavy chain linked by a single disulfide bond between cysteine 132 and cysteine 289 (S. Bajaj et al., Biochemistry 22, 4047 (1983)).

[00146] The structural organization of FIX is similar to that of the vitamin K-dependent blood clotting proteins FVII, FX and protein C (B. Furie and B. Furie, supra). The approximately 45 amino acids of the amino terminus comprise the gamma-carboxyglutamic acid, or Gla, domain. This is followed by two epidermal growth factor homology domains (EGF), an activation peptide and the catalytic "heavy chain" which is a member of the serine protease family (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993); S. Spitzer et al., Biochemical Journal 265, 219 (1990); H. Brandstetter et al., Proc. Natl. Acad Sci. USA 92, 9796 (1995)).

[00147] "Equivalent dose," as used herein, means the same dose of FVIII activity as expressed in International Units, which is independent of molecular weight of the polypeptide in question. One International Unit (IU) of FVIII activity corresponds approximately to the quantity of FVIII in one milliliter of normal human plasma. Several assays are available for measuring FVIII activity, including the European Pharmacopoeia chromogenic substrate assay and a one stage clotting assay.

[00148] "Fc," as used herein, means functional neonatal Fc receptor (FcRn) binding partners, unless otherwise specified. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. Thus, the term Fe includes any variants of IgG Fc that are functional. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., Nature 372:379 (1994), incorporated herein by reference in its entirety). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include, e.g., whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain.

[00149] References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U. S. Department of Public Health, Bethesda; MD, incorporated herein by reference in its entirety. (The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp. Med. 180: 2377 (1994), incorporated herein by reference in its entirety.) An Fc can comprise the CH2 and CH3 domains of an immunoglobulin with or without the hinge region of the immunoglobulin. Exemplary Fc variants are provided in WO 2004/101740 and WO 2006/074199, incorporated herein by reference in its entirety.

[00150] An Fc (or Fc portion of a chimeric polypeptide) can contain one or more mutations, and combinations of mutations. E.g., an Fc (or Fc portion of a chimeric polypeptide) can contain mutations conferring increased half-life such as M252Y, S254T, T256E, and combinations thereof, as disclosed in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which is incorporated herein by reference in its entirety; H433K, N434F, and combinations thereof, as disclosed in Vaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporated herein by reference in its entirety; the mutants disclosed at pages 1-2, paragraph [0012], and Examples 9 and 10 of U.S. Application Publ. No. 2009/0264627 Al, which is incorporated herein by reference in its entirety; and the mutants disclosed at page 2, paragraphs [0014] to [0021] of U.S. Application Publ. No. 20090163699 Al, which is incorporated herein by reference in its entirety. 1001511 Fc (or Fc portion of a chimeric polypeptide) can also include, e.g., the following mutations: The Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include, e.g., modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example the following single amino acid residues in human IgGI Fc (Fcyl) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F,

R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wildtype proline substituted by alanine at position number 238. In addition to alanine other amino acids can be substituted for the wildtype amino acids at the positions specified above.

[00152] Mutations can be introduced singly into Fc giving rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations can be introduced together, giving rise to hundreds more FcRn binding partners. Certain of these mutations can confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcyRl, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847, which is incorporated herein by reference in its entirety; Friend et al. 1999, Transplantation 68:1632, which is incorporated herein by reference in its entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, which is incorporated herein by reference in its entirety). 100153] Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity can arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgGI "ELLG" to the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRII which mediate various effector functions will not bind to IgGI when such mutations have been introduced (Ward and Ghetie, Therapeutic Immunology 2:77 (1995), which is incorporated herein by reference in its entirety; and Armour et al., Eur. J. Immunol. 29:2613 (1999), which is incorporated herein by reference in its entirety). As a further example of new functionality arising from mutations described above affinity for FcRn can be increased beyond that of wild type in some instances. This increased affinity can reflect an increased "on" rate, a decreased "off' rate or both an increased "on" rate and a decreased "off' rate. Mutations believed to impart an increased affinity for FcRn include, e.g., T256A, T307A, E380A, and N434A (Shields et al., J. Biol. Chem. 276:6591 (2001), which is incorporated herein by reference in its entirety).

[00154] The Fc (or Fc portion of a chimeric polypeptide) can be at least 90% or 95% identical to the Fc amino acid sequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6). The Fc (or Fc portion of a chimeric polypeptide) can be identical to the Fc amino acid sequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 and amino acids 2352 to 2578 of SEQ ID NO:6).

[00155] "Hybrid" polypeptides and proteins, as used herein, means a combination of a chimeric polypeptide with a second polypeptide. The chimeric polypeptide and the second polypeptide in a hybrid can be associated with each other via protein-protein interactions, such as charge-charge or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in a hybrid can be associated with each other via disulfide or other covalent bond(s). Hybrids are described in WO 2004/101740 and WO 2006/074199, each of which is incorporated herein by reference in its entirety. See also U.S. Patent Nos. 7,404,956 and 7,348,004, each of which is incorporated herein by reference in its entirety. The second polypeptide can be a second copy of the same chimeric polypeptide or it can be a non-identical chimeric polypeptide. See, e.g., FIG. 1, Example 1, and TABLE 2.

[00156] In one embodiment, the second polypeptide is a polypeptide comprising an Fc. In another embodiment, the chimeric polypeptide is a chimeric FVIII-Fc polypeptide and the second polypeptide consists essentially of Fc, e.g., the hybrid polypeptide of Example 1, which is a rFVIIIFc recombinant fusion protein consisting of a single molecule of recombinant B-domain deleted human FVIII (BDD-rFVIII) fused to the dimeric Fc domain of the human IgG1, with no intervening linker sequence. This hybrid polypeptide is referred to herein as FVIIIFc monomeric Fc fusion protein, FVIIIFc monomer hybrid, monomeric FVIIIIFc hybrid, and FVIIIFc monomer-dimer. See Example 1, Fig. 1, and TABLE 2A. The Examples provide preclinical and clinical data for this hybrid polypeptide. 100157] The second polypeptide in a hybrid can comprise or consist essentially of a sequence at least 90% or 95% identical to the amino acid sequence shown in TABLE 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or at least 90% or 95% identical to the amino acid sequence shown in TABLE 2A(ii) with a signal sequence (amino acids 1 to 247 of

SEQ ID NO-:4). The second polypeptide can comprise or consist essentially of a sequence identical to the amino acid sequence shown in TABLE 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or identical to the amino acid sequence shown in TABLE 2A(ii) with a signal sequence (amino acids 1 to 247 of SEQ ID NO:4).

[00158] FIG. 1 is a schematic showing the structure of a B domain deleted FVIII-Fc chimeric polypeptide, and its association with a second polypeptide that is an Fc polypeptide. To obtain this hybrid, the coding sequence of human recombinant B-domain deleted FVIII was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human liver poly A RNA (Clontech) using FVIII-specific primers. The FVIII sequence includes the native signal sequence for FVIII. The B-domain deletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638; 4969 bp) for a total deletion of 2682 bp. Then, the coding sequence for human recombinant Fc was obtained by RT-PCR from a human leukocyte cDNA library (Clontech) using Fc specific primers. Primers were designed such that the B-domain deleted FVIII sequence was fused directly to the N-terminus of the Fc sequence with no intervening linker. The FVIIIFc DNA sequence was cloned into the mammalian dual expression vector pBUDCE4.1 (Invitrogen) under control of the CMV promoter. A second identical Fc sequence including the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of the second promoter, EF a, in the expression vector pBUDCE4.1.

[00159] The rFVIIIFc expression vector was transfected into human embryonic kidney 293 cells (HEK293H; Invitrogen) using Lipofectamine 2000 transfection reagent (Invitrogen). Stable clonal cell lines were generated by selection with Zeocin (Invitrogen). One clonal cell line, 3C4 22 was used to generate FVIIIFc for characterization in vivo. Recombinant FVIIIFc was produced and purified (McCue et al. 2009) at Biogen Idec (Cambridge, MA). The transfection strategy described above was expected to yield three products, i.e., monomeric rFVIIIFc hybrids, dimeric rFVIIIFc hybrids and dimeric Fc. However, there was essentially no dimeric rFVIIIFc detected in the conditioned medium from these cells. Rather, the conditioned medium contained Fc and monomeric rFVIIIFc. It is possible that the size of dimeric rFVIIIFc was too great and prevented efficient secretion from the cell. This result was beneficial since it rendered the purification of the monomer less complicated than if all three proteins had been present. The material used in these studies had a specific activity of approximately 9000 IU/mg.

[00160] "Dosing interval," as used herein, means the dose of time that elapses between multiple doses being administered to a subject. The comparison of dosing interval can be carried out in a single subject or in a population of subjects and then the average obtained in the population can be calculated.

[00161] The dosing interval when administering a chimeric FVIII polypeptide, e.g., a chimeric FVIII-Fc polypeptide (a polypeptide comprising a FVIII or a hybrid) of the present disclosure can be at least about one and one-half times longer than the dosing interval required for an equivalent dose of said FVIII without the non-FVIII portion, e.g., without the Fc portion (a polypeptide consisting of said FVIII). The dosing interval can be at least about one and one-half to six times longer, one and one-half to five times longer, one and one-half to four times longer, one and one-half to three times longer, or one and one-half to two times longer, than the dosing interval required for an equivalent dose of said FVIII without the non-FVIII portion, e.g., without the Fc portion (a polypeptide consisting of said FVIII).

[00162] The dosing interval can be at least about one and one-half, two, two and one-half, three, three and one-half, four, four and one-half, five, five and one-half or six times longer than the dosing interval required for an equivalent dose of said FVIII without the non-FVIII portion, e.g., without the Fc portion (a polypeptide consisting of said FVIII).. The dosing interval can be about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer. The dosing interval can be at least about one and one-half to 5, one and one-half, 2, 3, 4, or 5 days or longer. For on-demand treatment, the dosing interval of said chimeric polypeptide or hybrid is about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or longer. 100163] In one embodiment, the effective dose is 25-65 IU/kg (25, 26, 27, 28, 29, 30, 31, 32, 33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58, 59, 60, 61, 62, 62, 64, or 65 IU/kg) and the dosing interval is once every 3-5, 3-6, 3-7, 3, 4, 5, 6, 7, or 8 or more days, or three times per week, or no more than three times per week. In another embodiment, the effective dose is 65 IU/kg and the dosing interval is once weekly, or once every 6-7 days. The doses can be administered repeatedly as long as they are necessary (e.g., at least 10, 20, 28, 30, 40, 50, 52, or 57 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). 100164] In certain embodiments, the effective dose for on-demand treatment is 20-50 IU/Kg (20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, 46, 47, 48, 49, or 50 IU/kg). The on-demand treatment can be one time dosing or repeated dosing. For repeated dosing, the dosing interval can be every 12-24 hours, every 24-36 hours, every 24-48 hours, every 36-48 hours, or every 48-72 hours. Accordingly, the term "repeated dosing" refers to the administration of more than one dose over a period of time. Doses administered under a "repeated dosing" regimen are referred to as "repeated doses." In some embodiments, each of the repeated doses is separated from another by at least about 12 hours, at least about 24 hours, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, at least about ten days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days. In some embodiments, the repeated doses comprise at least about two doses, at least about five doses, at least about 10 doses, at least about 20 doses, at least about 25 doses, at least about 30 doses, at least about 35 doses, at least about 40 doses, at least about 45 doses, at least about 50 doses, at least about 55 doses, at least about 60 doses, at least about 65 doses, or at least about 70 doses. the repeated doses comprise from about two doses to about 100 doses, from about five doses to about 80 doses, from about 10 doses to about 70 doses, from about 10 doses to about 60 doses, from about 10 doses to about 50 doses, from about 15 doses to about 40 doses, from about 15 doses to about 30 doses, from about 20 doses to about 30 doses, or from about 20 doses to about 40 doses. the repeated doses comprise about two doses, about five doses, about 10 doses, about 15 doses, about 20 doses, about 25 doses, about 30 doses, about 35 doses, about 40 doses, about 45 doses, about 50 doses, about 55 doses, about 60 doses, about 65 does, about 70 doses, about 75 doses, about 80 doses, about 90 doses, or about 100 doses.

[00165] In some embodiments, the subject is further administered, after the repeated doses, a pharmaceutical composition comprising a clotting factor protein which comprises the clotting factor, but does not comprise an Fc portion. This clotting factor can be a full length or mature clotting factor. In some embodiments, such clotting factor can be ADVATE@, RECOMBINATE@, KOGENATE FS@, HELIXATE FS@, XYNTHA@/REFACTO AB@, HEMOFIL-M@, MONARC-M@, MONOCLATE-P@, HUMATE-P@, ALPHANATE@, KOATE-DVI@, AND HYATE:C@

[00166] The terms "long-acting" and "long-lasting" are used interchangeably herein. "Long-lasting clotting factors" or "long-acting clotting factors" (e.g., long-acting FVII, long acting FVIII, and long-acting FIX) are clotting factors, e.g., FVII, FVIII, or FIX, having an increased half-life (also referred to herein as ti/2 , t1 /2 beta, elimination half-life and HL) over a reference clotting factor, e.g., FVII, FVIII or FIX. The "longer" FVIII activity can be measured by any known methods in the art, e.g., aPTT assay, chromogenic assay, ROTEM, TGA, and etc. In one embodiment, the "longer" FVIII activity can be shown by the T 2beta (activity). In another embodiment, the "longer" FVIII activity can be shown by the level of FVIII antigen present in plasma, e.g., by the T/ 2beta (antigen). In other embodiments, the long-acting or long lasting FVIII polypeptide works longer in a coagulation cascade, e.g., is active for a longer period, compared to a wild-type FVIII polypeptide, REFACTO* or ADVATE". The increased half-life of a long-acting clotting factor, e.g., a long-acting FVIII, can be due to fusion to one or more non-clotting factor polypeptides such as, e.g., Fc. The increased half-life can be due to one or more modification, such as, e.g., pegylation. Exemplary long-acting clotting factor (e.g., long-acting FVIII polypeptides) include, e.g., chimeric clotting factors (e.g., chimeric FVIII polypeptides comprising Fc), and chimeric clotting factors comprising albumin (e.g., chimeric FVIII polypeptides comprising albumin). Additional exemplary long-acting clotting factors (e.g., long-acting FVIII polypeptides) include, e.g., pegylated clotting factors (e.g., pegylated FVIII). 100167] The "reference" polypeptide, e.g., in the case of a long-acting chimeric clotting factor (e.g., a long-acting chimeric FVIII polypeptide), is a polypeptide consisting essentially of the clotting factor portion of the chimeric polypeptide. For example, in the case of a long-acting FVIII, the reference polypeptide is the FVIII portion of the chimeric polypeptide, e.g., the same FVIII portion without the Fc portion, or without the albumin portion. Likewise, the reference polypeptide in the case of a modified FVIII is the same FVIII without the modification, e.g., a FVIII without the pegylation. 100168] In some embodiments, the long-acting FVIII has one or more of the following properties when administered to a subject: a mean residence time (MRT) (activity) in said subject of about 14-41.3 hours; a clearance (CL) (activity) in said subject of about 1.22-5.19 mL/hour/kg or less; a t1 2beta (activity) in said subject of about 11-26.4 hours; an incremental recovery (K value) (activity; observed) in said subject of about 1.38-2.88 IU/dL per IU/kg; a V,, (activity) in said subject of about 37.7-79.4 mL/kg; and an AUC/dose in said subject of about 19.2-81.7 IU*h/dL per IU/kg.

[00169] In some embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: a mean incremental recovery (K-Value) (activity; observed) greater that 1.38 IU/dL per IU/kg; a mean incremental recovery (K-Value) (activity; observed) of at least about 1.5, at least about 1.85, or at least about 2.46 IU/dL per IU/kg. a mean clearance (CL) (activity) in said patient population of about 2.33 ±1.08 mL/hour/kg or less; a mean clearance (CL) (activity) in said patient population of about 1.8-2.69 mL/hour/kg; a mean clearance (CL) (activity) in said patient population that is about 65% of the clearance of a polypeptide comprising said FVIII without modification; a mean mean residence time (MRT) (activity) in said patient population of at least about 26.3 ±8.33 hours; a mean MRT (activity) in said patient population of about 25.9 - 26.5 hours; a mean MRT (activity) in said patent population that is about 1.5 fold longer than the mean MRT of a polypeptide comprising said FVIII without modification; a mean t1 /2beta (activity) in said patient population of about 18.3 ±5.79 hours; a mean t1 /2beta (activity)in said patient population that is about 18 - 18.4 hours; a mean t1 /2beta (activity) in said patient population that is about 1.5 fold longer than the mean ti/2beta of a polypeptide comprising said FVIII without modification; a mean incremental recovery (K value) (activity; observed) in said patient population of about 2.01 0.44 IU/dL per IU/kg; a mean incremental recovery (K value) (activity; observed) in said patient population of about 1.85 - 2.46 IU/dL per IU/kg; a mean incremental recovery (K value) (activity; observed) in said patient population that is about 90 % of the mean incremental recovery of a polypeptide comprising said FVIII without modification; a mean Vss (activity) in said patient population of about 55.1 ±12.3 mL/kg; a mean Vss (activity) in said patient population of about 45.3 - 56.1 mL/kg; a mean AUC/dose (activity) in said patient population of about 49.9 ±18.2 IU*h/dL per IU/kg; a mean AUC/dose (activity) in said patient population of about 44.8 - 57.6 IU*h/dL per IU/kg.

100170] In other embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: a CmaxOBS in said subject administered with the chimeric polypeptide is comparable to the Cmax_OBS in a subject administered with the same amount of a polypeptide consisting of the full-length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a Cmax_OBS in said subject of about 60.5 IU/dL, about 60.5 ±1 IU/dL, about 60.5 2 IU/dL, about 60.5± 3 IU/dL, about 60.5 4 IU/dL, about 60.5 5 IU/dL, about 60.5± 6 IU/dL, about 60.5 7 IU/dL, about 60.5 8 IU/dL, about 60.5 9 IU/dL, or about 60.5 ±10 IU/dL as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a Cmax_OBS in said subject of about 53.1 - 69 IU/dL as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a CmaxOBS in said subject of about 119 IU/dL, about 119 ±1 IU/dL, about 119 2 IU/dL, about 119 3 IU/dL, about 119± 4 IU/dL, about 119± 5 IU/dL, about 119± 6 IU/dL, about 119± 7 IU/dL, about 119± 8 IU/dL, about 119± 9 IU/dL, about 119 ±10 IU/dL, about 119 11 IU/dL, about 119 ±12 IU/dL, about 119 13 IU/dL, about 119 14 IU/dL, about 119 ±15 IU/dL, about 119 16 IU/dL, about 119 ±17 IU/dL, or about 119 ±18 IU/dL, as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; a Cmax_OBS in said subject of about 103 - 136 IU/dL as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; aCmax_OBS in said subject of about 76.5 IU/dL, about 76.5 ±1 IU/dL, about 76.5 2 IU/dL, about 76.5± 3 IU/dL, about 76.5 4 IU/dL, about 76.5 5 IU/dL, about 76.5± 6 IU/dL, about 76.5± 7 IU/dL, about 76.5 8 IU/dL, about 76.5± 9 IU/dL, about 76.5 10 IU/dL, about 76.5 11 IU/dL, about 76.5 12 IU/dL, about 76.5 13 IU/dL, about 76.5 ±14 IU/dL, or about 76.5 ±15 IU/dL, as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a Cmax_OBS in said subject of about 64.9 - 90.1 IU/dL as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a Cmax_OBS in said subject of about 182 IU/dL, about 182±2 IU/dL, about 182± 4 IU/dL, about 182± 6 IU/dL, about 182± 8 IU/dL, about 182 ±10 IU/dL, about 182 ±12 IU/dL, about 182 ±14 IU/dL, about 182 ±16 IU/dL, about 182 ±18 IU/dL, or about 182 ±20 IU/dL as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered; or a CmaOBS in said subject of about 146 - 227 IU/dL, about 146± 5 IU/dL, about 146 10 IU/dL, about 227± 5 IU/dL, or about 146± 10 IU/dL as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered.

[00171] In certain embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: a t1 2beta (activity) in said subject that is at least 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, or 1.90 times higher than the t 2beta (activity) in a subject administered with the same amount of a polypeptide consisting of the full-length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a t1 2beta (activity) in said subject of about 18.8 hours, 18.8 1 hours, 18.8 ±1 hours, 18.8± 2 hours, 18.8± 3 hours, 18.8 4 hours, 18.8 5 hours, 18.8 6 hours, 18.8± 7 hours, 18.8 8 hours, 18.8± 9 hours, 18.8 10 hours, or 18.8 ±11 hours as measured by a one stage (aPTT) assay; a t1 2beta (activity) in said subject of about 14.3 - 24.5 hours as measured by a one stage (aPTT) assay; a t1 2beta (activity) in said subject of about 16.7 hours, 16.7 1 hours, 16.7± 2 hours, 16.7± 3 hours, 16.7± 4 hours, 16.7 5 hours, 16.7 6 hours, 16.7 7 hours, 16.7± 8 hours, 16.7± 9 hours, 16.7 ±10 hours, or 16.7 ±11 hours as measured by a two stage (chromogenic) assay; a t/ 2beta (activity) in said subject of about 13.8 - 20.1 hours as measured by a two stage (chromogenic) assay; a t1 2beta (activity) in said subject of about 19.8 hours, 19.8 1 hours, 19.8± 2 hours, 19.8± 3 hours, 19.8± 4 hours, 19.8± 5 hours, 19.8± 6 hours, 19.8 7 hours, 19.8± 8 hours, 19.8± 9 hours, 19.8 ±10 hours, or 19.8±11 hours as measured by a two stage (chromogenic) assay; or a t1 2beta (activity) in said subject of about 14.3 - 27.5 hours as measured by a two stage (chromogenic) assay.

100172] In certain embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: a clearance (CL) (activity) in said subject is 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, or 0.70 times lower than the clearance in a subject administered with the same amount of a polypeptide consisting of the full length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a clearance (CL) (activity) in said subject of about 1.68 mL/hour/kg, 1.68 0.1 mL/hour/kg, 1.68 0.2 mL/hour/kg, 1.68 ±0.3 mL/hour/kg, 1.68 0.4 mL/hour/kg, 1.68 0.5 mL/hour/kg, 1.68 0.6 mL/hour/kg, or 1.68 0.7 mL/hour/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.31 - 2.15 mL/hour/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 2.32 mL/hour/kg, 2.32 0.1 mL/hour/kg, 2.32 0.2 mL/hour/kg, 2.32 ±0.3 mL/hour/kg, 2.32 0.4 mL/hour/kg, 2.32 0.5 mL/hour/kg, 2.32 0.6 mL/hour/kg, or 2.32 0.7 mL/hour/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.64 - 3.29 mL/hour/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.49 mL/hour/kg, 1.49 0.1 mL/hour/kg, 1.49 0.2 mL/hour/kg, 1.49 ±0.3 mL/hour/kg, 1.49 0.4 mL/hour/kg, 1.49 0.5 mL/hour/kg, 1.49 0.6 mL/hour/kg, or 1.49 ±0.7 mL/hour/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.16 - 1.92 mL/hour/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.52 mL/hour/kg, 1.52 0.1 mL/hour/kg, 1.52 0.2 mL/hour/kg, 1.52 ±0.3 mL/hour/kg, 1.52 0.4 mL/hour/kg, 1.52 0.5 mL/hour/kg, 1.52 0.6 mL/hour/kg, or 1.52 0.7 mL/hour/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered; or a clearance (CL) (activity) in said subject of about 1.05-2.20 mL/hour/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered.

[00173] In some embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: a MRT in said subject is at least 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, or 1.93 times higher than the MRT in a subject administered with the same amount of a polypeptide consisting of the full-length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a MRT (activity) in said subject of about 27 hours, 27 1 hours, 27± 2 hours, 27 3 hours, 27± 4 hours, 27± 5 hours, 27 6 hours, 27 7 hours, 27± 8 hours, 27 9 hours, or 27 10 hours as measured by a one stage (aPTT) assay; a MRT (activity) in said subject of about 20.6 - 35.3 hours as measured by a one stage (aPTT) assay; a MRT (activity) in said subject of about 23.9 - 28.5 hours as measured by a two stage (chromogenic) assay; a MRT (activity) in said subject of about 19.8 - 28.9 hours as measured by a two stage (chromogenic) assay; or a MRT (activity) in said subject of about 20.5 - 39.6 hours as measured by a two stage (chromogenic) assay. 1001741 In other embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: an incremental recovery in said subject that is comparable to the Incremental Recovery in a subject administered with the same amount of a polypeptide consisting of the full-length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; an incremental recovery in said subject of about 2.44 IU/dL per IU/kg, 2.44 0.1 IU/dL per IU/kg, 2.44 ±0.2 IU/dL per IU/kg, 2.44 ±0.3 IU/dL per IU/kg, 2.44 ±0.4 IU/dL per IU/kg, 2.44 ±0.5 IU/dL per IU/kg, 2.44 ±0.6 IU/dL per IU/kg, 2.44 ±0.7 IU/dL per IU/kg, 2.44 ±0.8 IU/dL per IU/kg, 2.44 ±0.9 IU/dL per IU/kg, 2.44 ±1.0 IU/dL per IU/kg, 2.44 ±1.1 IU/dL per

IU/kg, or 2.44 ±1.2 IU/dL per1U/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 2.12 - 2.81 IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 1.83 IU/dL per IU/kg, 1.83 0.1 IU/dL per IU/kg, 1.83 ±0.2 IU/dL per IU/kg, 1.83 ±0.3 IU/dL per IU/kg, 1.83 ±0.4 IU/dL per IU/kg, 1.83 ±0.5 IU/dL per IU/kg, 1.83 ±0.6 IU/dL per IU/kg, 1.83 0.7 IU/dL per IU/kg, 1.83 0.8 IU/dL per IU/kg, 1.83 0.9 IU/dL per IU/kg, 1.83 ±1.0 IU/dL per IU/kg, or 1.83 ±1.1 IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 1.59 - 2.10 IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 3.09 IU/dL per IU/kg, 3.09 0.1 IU/dL per IU/kg, 3.09 0.2 IU/dL per IU/kg, 3.09 ±0.3 IU/dL per IU/kg, 3.09 0.4 IU/dL perrU/kg, 3.09 ±0.5 IU/dL per IU/kg, 3.09 ±0.6 IU/dL per IU/kg, 3.09 0.7 IU/dL per IU/kg, 3.09 0.8 IU/dL per IU/kg, 3.09 ±0.9 IU/dL per IU/kg, 3.09 ±1.0 IU/dL per IU/kg, 3.09 ±1.1 IU/dL per IU/kg, 3.09 ±1.2 IU/dL per rU/kg, or 3.09 ±1.3 IU/dL per IU/kg, as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 2.80 IU/dL per IU/kg, 2.80 0.1 IU/dL per IU/kg, 2.80 ±0.2 IU/dL per IU/kg, 2.80 ±0.3 IU/dL per IU/kg, 2.80 ±0.4 IU/dL per IU/kg, 2.80 ±0.5 IU/dL per IU/kg, 2.80 ±0.6 IU/dL per IU/kg, 2.80 0.7 IU/dL per IU/kg, 2.80 0.8 IU/dL per IU/kg, 2.80 ±0.9 IU/dL per IU/kg, 2.80 ±1.0 IU/dL per IU/kg, 2.80 ±1.1 IU/dL per IU/kg, or 2.80 ±1.2 IU/dL per IU/kg, as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 2.61-3.66 IU/dL per IU/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; or an incremental recovery in said subject of about 2.24-3.50 IU/dL per IU/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered.

[00175] In still other embodiments, the long-acting FVIII has one or more of the following properties when administered to a patient population: a Vs, (activity) in said subject that is comparable to the Vss (activity) in a subject administered with the same amount of a polypeptide consisting of the full-length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a V,, (activity) in said subject of about 45.5 mL/kg, 45.5 1 mL/kg, 45.5± 2 mL/kg, 45.5 3 mL/kg, 45.5 4 mL/kg, 45.5 + 5 mL/kg, 45.5± 6 mL/kg, 45.5 7 mL/kg, 45.5 8 mL/kg, 45.5 9 mL/kg, 45.5 10 mL/kg, or 45.5 11 mL/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a Vss (activity) in said subject of about 39.3 - 52.5 nL/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a V,, (activity) in said subject of about 62.8 mL/kg, 62.8 ±1 mL/kg, 62.8 2 mL/kg, 62.8 3 mL/kg, 62.8 4 mL/kg, 62.8± 5 mL/kg, 62.8 6 mL/kg, 62.8 7 mL/kg, 62.8 8 mL/kg, 62.8± 9 mL/kg, 62.8 ±10 mL/kg, 62.8 ±11 mL/kg, 62.8 ±12 mL/kg, 62.8 + 13 mL/kg, 62.8± 14 mL/kg, 62.8 ±15 mL/kg, or 62.8 + 16 mL/kg as measured by a one stage (aPTT) assay when about 65 lU/kg of the chimeric polypeptide is administered; a Vss (activity) in said subject of about 55.2 - 71.5 mL/kg as measured by a one stage (aPTT) assay when about 65 lU/kg of the chimeric polypeptide is administered; a V,, (activity) in said subject of about 35.9 mL/kg, 35.9 1 mL/kg, 35.9± 2 mL/kg, 35.9 3 mL/kg, 35.9 4 mL/kg, 35.9 ± 5 mL/kg, 35.9 6 mL/kg, 35.9 + 7 mL/kg, 35.9 8 mL/kg, 35.9 9 mL/kg, 35.9 + 10 mL/kg, 35.9 ±11 mL/kg, 35.9 12 mL/kg, or 35.9 + 13 mL/kg, as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a V,, (activity) in said subject of about 30.4-42.3 mL/kg as measured by a two stage (chromogenic) assay when about 25 lU/kg of the chimeric polypeptide is administered; a V,, (activity) in said subject of about 43.4 mL/kg, 43.4 + 1 mL/kg, 43.4± 2 mL/kg, 43.4 3 mL/kg, 43.4 + 4 mL/kg, 43.4 5 mL/kg, 43.4 6 mL/kg, 43.4 7 mL/kg, 43.4 + 8 mL/kg, 43.4 9 mL/kg, 43.4 10 mL/kg, 43.4 + 11 mL/kg, 43.4 12 mL/kg, 43.4 13 mL/kg, 43.4± 14 mL/kg, 43.4 15 mL/kg, or 43.4 16mL/kg, as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered; or a V,, (activity) in said subject of about 38.2-49.2 mL/kg as measured by a two stage (chromogenic) assay when about 65 RU/kg of the chimeric polypeptide is administered.

[00176) In yet other embodiments, the long-acting FVII has one or more of the following properties when administered to a patient population: an AUCINF in said subject that is at least 1.45 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90 times higher than the AUCfNF in a subject administered with the same amount of a polypeptide consisting of the full-length, mature FVIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; an AUCINF in said subject of about 1440 +316 hr*IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1160 - 1880 hr*IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1480 hr*IU/dL per IU/kg, 1480 + 100 hr*IU/dL per IU/kg, 1480 ±200 hr*IU/dL per IU/kg, 1480 300 hr*IU/dL per IU/kg, 1480 400 hr*IU/dL per IU/kg, 1480 + 500 hr*IU/dL per IU/kg, 1480 600 hr*IU/dL per IU/kg, 1480 700 hr*IU/dL per IU/kg, 1480 800 hr*IU/dL per IU/kg, 1480 900 hr*IU/dL per IU/kg, or 1480+ 1000 hr*IU/dL per IU/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 2910 + 1320 hr*IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1980 - 3970 hr*IU/dL per lU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 2800 hr*IU/dL per IU/kg, 2800 + 100 hr*IU/dL per IU/kg, 2800 200 hr*IU/dL per IU/kg, 2800 ±300 hr*IU/dL per IU/kg, 2800 400 hr*IU/dL per lU/kg, 2800 500 hr*IU/dL per IU/kg, 2800 + 600 hr*IU/dL per IU/kg, 2800 700 hr*IU/dL per IU/kg, 2800 800 hr*IU/dL per IU/kg, 2800 900 hr*IU/dL per lU/kg, or 2800+ 1000 hr*IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an AUCNF in said subject of about 1660 hr*IU/dL per IU/kg, 1660 100 hr*IU/dL per IU/kg, 1660 200 hr*IU/dL per IU/kg, 1660 300 hr*IU/dL per IU/kg, 1660 400 hr*IU/dL per IU/kg, 1660 500 hr*IU/dL per IU/kg, 1660 600 hr*IU/dL per IU/kg, 1660 700 hr*IU/ 6/kg, 1660 800 hr*IU/dL per IU/kg, 1660+ 900 hr*IU/dL per IU/kg, or 1660± 1000 hr*IU/dL per IU/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1300 - 2120 hr*IU/dL per LU/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 4280 hr*IU/dL per IU/kg, 4280 100 hr*IU/dL per IU/kg, 4280 200 hr*IU/dL per IU/kg, 4280 300 hr*IU/dL per IU/kg, 4280 400 hr*IU/dL per IU/kg, 4280± 500 hr*IU/dL per IU/kg, 4280± 600 hr*IU/dL per IU/kg, 4280 ± 700 hr*IU/dL per IU/kg, 4280 800 hr*IU/dL per IU/kg, 4280 900 hr*IU/dL per IU/kg, 4280± 1000 hr*IU/dL per IU/kg, 4280 ± 1100 hr*IU/dL per IU/kg, 4280 1200 hr*IU/dL per IU/kg, 4280 1300 hr*IU/dL per IU/kg, 4280 ± 1400 hr*IU/dL per IU/kg, 4280 1500 hr*IU/dL per IU/kg, or 4280 1600 hr*IU/dL per IU/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered; or an AUCINF in said subject of about 2960 - 6190 hr*IU/dL per IlU/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered.

[00177] "On-demand treatment," as used herein, means treatment that is intended to take place over a short course of time and is in response to an existing condition, such as a bleeding episode, or a perceived need such as planned surgery. The terms "on-demand treatment" and "episodic treatment" are used interchangeably. Conditions that can require on-demand (episodic) treatment include, e.g., a bleeding episode, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. The subject can be in need of surgical prophylaxis, peri-operative management, or treatment for surgery. Such surgeries include, e.g., minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

[001781 In one embodiment, on-demand (episodic) treatment resolves greater than 80% (greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% of bleeds (e.g., spontaneous bleeds) in a single dose. In another embodiment, greater than 80% (greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than

91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% of bleeding episodes are rated excellent or good by physicians after on demand (episodic) treatment. In other embodiments, greater than 5%, (greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20%), or 5-20%, 5-15%, 5-10%, 10-20%, or 10-15% of bleeding episodes are rated as fair by physicians after on demand (episodic) treatment.

[001791 "Polypeptide," "peptide" and "protein" are used interchangeably and refer to a polymeric compound comprised of covalently linked amino acid residues.

[001801 "Polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric compound comprised of covalently linked nucleotide residues. Polynucleotides can be DNA, cDNA, RNA, single stranded, or double stranded, vectors, plasmids, phage, or viruses. Polynucleotides include, e.g., those in TABLE 1, which encode the polypeptides of TABLE 2 (see TABLE 1). Polynucleotides also include, e.g., fragments of the polynucleotides of TABLE 1, e.g., those that encode fragments of the polypeptides of TABLE 2, such as the FVIII, Fe, signal sequence, 6His and other fragments of the polypeptides of TABLE 2.

[001811 "Prophylactic treatment," as used herein, means administering a FVIII polypeptide in multiple doses to a subject over a course of time to increase the level of FVIII activity in a subject's plasma. The increased level can be sufficient to decrease the incidence of spontaneous bleeding or to prevent bleeding, e.g., in the event of an unforeseen injury. During prophylactic treatment, the plasma protein level in the subject can not fall below the baseline level for that subject, or below the level of FVIII that characterizes severe hemophilia (<1 IU/dl [1%]).

[00182] In one embodiment, the prophylaxis regimen is "tailored" to the individual patient, for example, by determining PK data for each patient and administering FVIII of the present disclosure at a dosing interval that maintains a trough level of 1-3% FVIII activity. Adjustments can be made when a subject experiences unacceptable bleeding episodes defined as >2 spontaneous bleeding episodes over a rolling two-month period. In this case, adjustment will target trough levels of 3-5%. In another embodiment, prophylactic treatment results in prevention and control of bleeding, sustained control of bleeding, sustained protection from bleeding, and/or sustained benefit. Prophylaxis, e.g., sustained protection can be demonstrated by an increased AUC to last measured time point (AUC-LAST) and reduced clearance, resulting in increased terminal tl/2 compared to short acting FVIII. Prophylaxis can be demonstrated by better Cm,, better Tm., and/or greater mean residence time versus short-acting FVIII. In some embodiments, prophylaxis results in no spontaneous bleeding episodes within about 24, 36, 48, 72, or 96 hours (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55, 56,57, 58,59, 60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80, 81,82, 83,84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours), after injection (e.g., the last injection). In certain embodiments, prophylaxis results in greater than 30% (e.g., greater than 31, 32, 33, 34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58, 59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83, 84, 85, 96, 87, 88, 89, or 90%, for example, greater than 50%), mean reduction in annualized bleeding episodes with once weekly dosing (e.g., at 65 IU/kg).

[00183] "Subject," as used herein means a human individual. Subject can be a patient who is currently suffering from a bleeding disorder or is expected to be in need of such a treatment. In some embodiments, the subject has never been previously treated with the clotting factor (i.e., the subject is a previously untreated subject or previously untreated patient). In some embodiments, the subject is a fetus and the methods comprises administering the chimeric polypeptide to the mother of the fetus and the administration to the subject occurs from the mother across the placenta. In some embodiments, the subject is a child or an adult. In some embodiments, the subject is a child less than one-year-old, less than two-year-old, less than three-year-old, less than four-year-old, less than five-year-old, less than six-year-old, less than seven-year-old, less than eight-year-old, less than nine-year-old, less than ten-year-old, less than eleven-year-old, or less than twelve-year-old. In some embodiments, the child is less than one-year old. In some embodiments, the child or adult subject develops a bleeding disorder, wherein the onset of the symptoms of the bleeding disorder is after the one-year-old age. In some embodiments, the administration of the chimeric polypeptide to the subject is sufficient to prevent, inhibit, or reduce development of an immune response selected from a humoral immune response, a cell-mediated immune response, or both a humoral immune response and a cell-mediated immune response against the clotting factor.

[00184] In some embodiments described herein, the subject is at risk of developing an inhibitory FVIII immune response. A subject can be at risk of developing an inhibitory FVIII immune response because, for example, the subject previously developed an inhibitory FVIII immune response or currently has an inhibitory FVIII immune response. In some embodiments, the subject developed an inhibitory FVIII immune response after treatment with a plasma-derived FVIII product. In some embodiments, the subject developed an inhibitory FVIII immune response after treatment with a recombinant FVIII product. In some embodiments, the subject developed an inhibitory FVIII immune response after treatment with a full-length FVIII protein. In some embodiments, the subject developed an inhibitory FVIII immune response after treatment with FVIII protein containing a deletion, e.g., a full or partial deletion of the B domain.

[00185] In some embodiments, the subject developed an inhibitory FVIII immune response after treatment with a FVIII product selected from the group consisting of ADVATE, RECOMBINATE*, KOGENATE FS® HELIXATE FS*, XYNTHA*/REFACTO AB, HEMOFIL-M®, MONARC-M*, MONOCLATE-P*, HUMATE-P®, ALPHANATE*, KOATE-DVI AND HYATE:C*.

[001861 In some embodiments, the immune response after treatment with a clotting factor, e.g., FVIII, comprises production of inhibitory antibodies to the clotting factor. In some embodiments, the inhibitory antibody concentration is at least 0.6 Bethesda Units (BU). In some embodiments, the inhibitory antibody concentration is at least 5 BU. In some embodiments, the inhibitory antibody concentration is at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8,0, at least 9.0, or at least 10.0 BU.

[00187] In some embodiments, the immune response after treatment with a clotting factor, e.g., FVIII, comprises a cell-mediated immune response. In some embodiments, the immune response comprises a cell-mediated immune response. The cell-mediated immune response can comprise that release of a cytokine selected from the group consisting of IL-12, IL-4 and TNF-a.

[00188] In some embodiments, the immune response after treatment with a clotting factor, e.g., FVIII, comprises a clinical symptom selected from the group consisting of: increased bleeding tendency, high clotting factor consumption, lack of response to clotting factor therapy, decrease efficacy of clotting factor therapy, and shortened half-life of clotting factor. In some embodiments, the subject has a mutation or deletion in the clotting factor gene. In some embodiments, the subject has a rearrangement in the the clotting factor gene. In some embodiments, the subject has severe hemophilia. In some embodiments, the subject has a relative who has previously developed an inhibitory immune response against the clotting factor. In some embodiments, the subject is receiving interferon therapy. In some embodiments, the subject is receiving antiviral therapy.

[00189] In some embodiments, the subject at risk has previously developed an inhibitory immune response to a therapeutic protein other than FVIII.

[001901 In addition, the subject can also be at risk of developing an inhibitory FVIII immune response as a result of a number of environmental or genetic factors that increase the likelihood the subject will develop an inhibitory immune response. Factors that increase the likelihood that a subject will develop an inhibitory immune response are known. See e.g., Kasper, C. "Diagnosis and Management of Inhibitors to Factors VIII and IX - An Introductory Discussion for Physicians," Treatment of Hemophilia 34 (2004), which is herein incorporated by reference in its entirety. For example, inhibitors arise more commonly in severe hemophilia (e.g., FVIII baseline level of less than 1%) than in mild or moderate hemophilia. See Report of Expert Meeting on FVIII Products and Inhibitor Development, European Medicines Agency (February 28, 2006-March 2, 2006), which is herein incorporated by reference in its entirety.

[00191] As a result of the fact that genetic factors can increase the likelihood that a subject will develop an inhibitory immune response, a subject with at least one family member (e.g., a sibling, parent, child, grandparent, aunt, uncle, or cousin) who has developed an inhibitory immune response to FVIII or another therapeutic protein can be at risk of developing an inhibitory FVIII immune response. 1001921 Inhibitors are common in patients with genetic mutations that prevent expression of FVIII such as large genetic deletions, nonsense mutations causing premature stop codons, and large inversions in the FVIII gene. Thus, in some embodiments, a subject at risk of developing an inhibitory FVIII immune response comprises a mutation, deletion, or rearrangement in a FVIII gene. In some embodiments, a subject at risk of developing an inhibitory FVIII immune response comprises a mutation in FVIII that prevents FVIII from binding to T cells. An association of inhibitors with large rearrangements of FVIII genes has been observed. See Astermark et al., Blood 107:3167-3172 (2006), which is herein incorporated by reference in its entirety. Thus, in some embodiments, a FVIII-Fc fusion protein is administered to a subject with a large rearrangement in a FVIII gene, for example, an intron 22 inversion or an intron 1 inversion. In some embodiments, FVIII-Fc is administered to a subject with two large rearrangements in FVIII. An association of inhibitors with null mutations has also been observed. Astermark et al, Blood 108:3739-3745 (2006). Thus, in some embodiments, a FVIII-Fc fusion protein is administered to a subject with a null mutation.

[00193] Inhibitors also arise more commonly after treatment with exogenous clotting factors on only a few occasions. Thus, in some embodiments, a subject at risk of developing an inhibitory immune response has had less than 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, or 5 exposure days. In some embodiments, a subject at risk of developing an inhibitory immune response has not been previously exposed to treatment with FVIII. 100194] In some embodiments, the subject is a fetus, Immune tolerance can be induced in a fetus by administering a chimeric polypeptide comprising a FVIII portion and an Fc portion to the mother of the fetus.

[001951 Inhibitors also arise more commonly in black African descent. See e-g, Kasper, C. "Diagnosis and Management of Inhibitors to Factors VIII and IX - An Introductory Discussion for Physicians," Treatment of Hemophilia 34 (2004), which is herein incorporated by reference in its entirety. Thus, in some embodiments, a subject at risk of developing an inhibitory immune response is of black African descent.

[00196] Genetic mutations in genes other than FVIII have also been linked with an increased risk of developing an inhibitory immune response. For example, the TNF- -308G>A polymorphism within Hap2, which is associated with increased constitutive and inducible transcription levels of TNF has been linked with an increased risk of developing an inhibitory immune response. See Astermark et al., Blood 108: 3739-3745 (2006), which is herein incorporated by reference in its entirety. Thus, in some embodiments, a subject at risk of developing an inhibitory immune response has a genetic polymorphism associated with increased TNF-c. In some embodiments, the polymorphism is the TNF- -308G>A polymorphism. In some embodiments, a subject at risk of developing an inhibitory immune response has a polymorphism in an IL10 gene, e.g. a polymorphism associated with increased secretion of IL10. In some embodiments, FVIII-Fc is administered to a subject with the allele 134 of the IL1OG microsatellite in the promote region of the IL10 gene. See Astermark et al. Hemostatis, Thrombosis, and Vascular Biology 108: 3739-3745 (2006), which is herein incorporated by reference in its entirety.

[00197] In some embodiments, a subject at risk of developing an inhibitory immune response has a genetic polymorphism associated with decreased CTLA-4 (Cytotoxic T-Lymphocyte

Antigen 4) expression. In some embodiments, a subject at risk of developing an inhibitory immune response has a mutation in DR15 (HLA-DR15) or DQB0602 MHC (Major histocompatibility complex) Class II molecules. Other MHC Class II molecules associated with the development of an inhibitory immune response in subjects with hemophilia are A3, B7, C7, DQA0102, C2, DQA0103, DQB0603, and DR13 (see Inhibitors in Patients with Hemophilia, E.C. Rodriguez-Merchan & C.A. Lee, Eds., Blackwell Science, Ltd,, 2002).

[00198] In some embodiments, the subject at risk for developing an inhibitory immune response has not been previously exposed to clotting factor, e.g., FVIII. In some embodiments, the subject at risk for developing an inhibitory immune response to a clotting factor, e.g., FVIII, has been exposed to FVIII. In some embodiments, the subject at risk for developing an inhibitory immune response to a clotting factor, e.g., FVIII, has had less than 150, less than 50, or less than 20 FVIII exposure days. In some embodiments, the subject has had at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days of clotting factor exposure. In some embodiments, the subject has had at least 25, 30, 35, 40, 45, or 50 days of clotting factor exposure. In some embodiments, the subject has had at least 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 days of clotting factor exposure.

[00199] In some embodiments, the inhibitory immune response is an inhibitory FVIII immune response. In some embodiments, the inhibitory FVIII immune response developed in response to a FVIII product selected from the group consisting of: ADVATE@, RECOMBINATE@, KOGENATE FS, HELIXATE FS@, XYNTHA@/REFACTO AB@, HEMOFIL-M@, MONARC-M@, MONOCLATE-P®, HUMATE-P@, ALPHANATE@, KOATE-DVI@, AND HYATE:C@. In some embodiments, the inhibitory immune response is an inhibitory FVIII immune response developed in response to a recombinant FVIII product. 1002001 In some embodiments, a subject at risk of developing an inhibitory immune response is receiving or has recently received an immunostimulatory therapy. For example, inhibitors have also been reported in HCV positive hemophilia A patients undergoing treatment with interferon as well as in HIV positive hemophilia A patients having an immune reconstitution inflammatory syndrome associated with anti-retroviral therapy. See Report of Expert Meeting on FVIII Products and Inhibitor Development, European Medicines Agency (February 28, 2006-March 2, 2006), which is herein incorporated by reference in its entirety. Thus, in some embodiments, a subject at risk of developing an inhibitory immune response is receiving interferon therapy. In some embodiments, a subject at risk of developing an inhibitory immune response is receiving an anti-retroviral therapy and having an immune reconstitution inflammatory syndrome.

[00201] An inhibitory FVIII immune response can be determined based on clinical responses to FVIII treatment. Clinical presentations of FVIII inhibitors are known and described, for example, in Kasper, C. "Diagnosis and Management of Inhibitors to Factors VIII and IX - An Introductory Discussion for Physicians," Treatment ofHemophilia 34 (2004), which is herein incorporated by reference in its entirety. For example, the presence of an inhibitor makes controlling hemorrhages for difficult, so immune responses to FVIII are signaled by an increased bleeding tendency, high FVIII consumption, lack of response to FVIII therapy, decreased efficacy of FVIII therapy, and/or shortened half-life of FVIII.

[00202] Inhibitory FVIIII immune responses can also be determined using laboratory tests such as the Bethesda test or the Nijmegan modification of the Bethesda test. A level of at least 0.6 Bethesda Units (BU) can indicate the presence of an inhibitory immune response. A level of at least 5 BU can indicate the presence of a high titer inhibitor. Measurements of the in vivo recovery and half-life of bolus FVIII infusion can also be used. 1002031 In some embodiments, a subject at risk of developing an inhibitory immune response has previously had an inhibitory immune response with a peak titer of at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0 BU.

[00204] In some embodiments provided herein, the methods comprise determining if a subject is at risk of developing an inhibitory FVIII immune response and administering to the subject a chimeric polypeptide comprising a FVIII portion and an Fe portion if the subject is at risk. Thus, in some embodiments, the methods comprise determining if the subject has a mutation, deletion, or rearrangement in FVIII and administering a chimeric polypeptide if the subject does. In some embodiments, the methods comprise deterring if the subject produces FVIII protein and administering a chimeric polypeptide if the subject does not. In some embodiments, the methods comprise determining if the subject has mild, moderate, or severe hemophilia, and administering a chimeric polypeptide if the subject has severe hemophilia. In some embodiments, the methods comprise determining if the subject has an inhibitory FVIII immune response, e.g., by assessing clinical manifestations of an immune response, measuring anti-FVIII antibody levels, titers, or activities, or measuring cell-mediated immune response (e.g., by measuring levels of cytokines) and administering a chimeric polypeptide if the subject has at least one of these indicators.

[00205] "Therapeutic dose," as used herein, means a dose that achieves a therapeutic goal, as described herein. The calculation of the required dosage of FVIII is based upon the empirical finding that, on average, 1 IU of FVIII per kg body weight raises the plasma FVIII activity by approximately 2 IU/dL. The required dosage is determined using the following formula: Required units = body weight (kg) x desired FVIII rise (IU/dL or % of normal) x 0.5 (IU/kg per IU/dL)

[00206] The therapeutic doses that can be used in the methods of the present disclosure are about 10-100 IU/kg, more specifically, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80 90, or 90-100 IU/kg, and more specifically, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg.

[00207] Additional therapeutic doses that can be used in the methods of the present disclosure are about 10 to about 150 IU/kg, more specifically, about 100-110, 110-120, 120-130, 130 140, 140-150 IU/kg, and more specifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU/kg.

[002081 "Variant," as used herein, refers to a polynucleotide or polypeptide differing from the original polynucleotide or polypeptide, but retaining essential properties thereof, e.g., FVIII coagulant activity or Fc (FcRn binding) activity. Generally, variants are overall closely similar, and, in many regions, identical to the original polynucleotide or polypeptide. Variants include, e.g., polypeptide and polynucleotide fragments, deletions, insertions, and modified versions of original polypeptides.

[00209] Variant polynucleotides can comprise, or alternatively consist of, a nucleotide sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide coding sequence in SEQ ID NO:1, 3, or 5 (the FVIII portion, the Fe portion, individually or together) or the complementary strand thereto, the nucleotide coding sequence of known mutant and recombinant FVIII or Fe such as those disclosed in the publications and patents cited herein or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:2, 4, or 6 (the FVIII portion, the Fe portion, individually or together), and/or polynucleotide fragments of any of these nucleic acid molecules (e-g., those fragments described herein). Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also included as variants, as are polypeptides encoded by these polynucleotides as long as they are functional.

[00210] Variant polypeptides can comprise, or alternatively consist of, an amino acid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NOS:2, 4, or 6 (the FVIII portion, the Fe portion, individually or together), and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).

[00211] By a nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. The query sequence can be, for example, the entire sequence shown in SEQ ID NO:1 or 3, the ORF (open reading frame), or any fragment specified as described herein.

[00212] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence or polypeptide of the present disclosure can be determined conventionally using known computer programs. In one embodiment, a method for determining the best overall match between a query sequence (reference or original sequence) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et aL, Comp. App. Biosci. 6:237-245 (1990), which is herein incorporated by reference in its entirety In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. In another embodiment, parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

[002131 If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present disclosure. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. 100214] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of the present disclosure.

[002151 By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present disclosure, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence can include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence can be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[002161 As a practical matter, whether any particular polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences of SEQ ID NO:2 (the FVIII portion, the Fc portion, individually or together) or 4, or a known FVIII or Fe polypeptide sequence, can be determined conventionally using known computer programs. In one embodiment, a method for determining the best overall match between a query sequence (reference or original sequence) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al, Comp. App. Biosci. 6:237-245(1990), incorporated herein by reference in its entirety. In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. In another embodiment, parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty-5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

100217] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present disclosure. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. 1002181 . For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of the present disclosure.

[00219] The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In one embodiment, the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In another embodiment, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. In other embodiments, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to others, e.g., a bacterial host such as E coli).

[002201 Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[00221] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or alter the characteristics of the polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al, J BioL Chem. 268: 2984-2988 (1993), incorporated herein by reference in its entirety, reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety.)

[00222] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) conducted extensive mutational analysis of human cytokine IL-la. They used random mutagenesis to generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." (See Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type. 100223] As stated above, polypeptide variants include, e.g., modified polypeptides. Modifications include, e.g., acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegyation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. In some embodiments, FVIII is modified, e.g., pegylated, at any convenient location. In some embodiments, FVIII is pegylated at a surface exposed amino acid of FVIII, e.g., a surface exposed cysteine, which can be an engineered cysteine. Id. In some embodiments, modified FVIII, e.g., pegylated FVIII, is a long-acting FVIII.

[00224] "Volume of distribution at steady state (Vss)," as used herein, has the same meaning as the term used in pharmacology, which is the apparent space (volume) into which a drug distributes. Vss = the amount of drug in the body divided by the plasma concentration at steady state.

[00225] "About," as used herein for a range, modifies both ends of the range. Thus, "about 10 20" means "about 10 to about 20."

[002261 The chimeric polypeptide used herein can comprise processed FVIII or single chain FVIII or a combination thereof. "Processed FVIII," as used herein means FVIII that has been cleaved at Arginine 1648 (for full-length FVIII) or Arginine 754 (for B-domain deleted FVIII), i.e., intracellular processing site. Due to the cleavage at the intracellular processing site, processed FVIII comprises two polypeptide chains, the first chain being a heavy chain and the second chain being a light chain. For example, the processed FVIII-Fc fusion protein (i.e., Heavy chain and Light chain fused to Fc) run at approximately 90 kDa and 130 kDa on a non-reducing SDS-PAGE, respectively, and 90 kDa and 105 kDa on a reducing SDS PAGE, respectively. Therefore, in one embodiment, at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the FVIII portion in the chimeric polypeptide is processed FVIII.

[00227 In another embodiment, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the FVIII portion in the chimeric polypeptide is processed FVIII In a particular embodiment, the chimeric polypeptide comprising processed FVIII is purified (or isolated) from the chimeric polypeptide comprising single chain FVIII, and at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the FVIII portion in the chimeric polypeptide is processed FVIII. 1002281 The terms "Single chain FVIII" or "SC FVIII" as used herein mean FVIII that has not been cleaved at the Arginine site (residue 1648 for full-length FVIII (i.e., residue 1667 of SEQ ID NO: 6) or residue 754 for B-domain deleted FVIII (i.e., residue 773 of SEQ ID NO: 2). Therefore, single chain FVIII in the chimeric polypeptide used herein comprises a single chain. In one embodiment, the single chain FVIII contains an intact intracellular processing site. The single chain FVIII-Fc fusion protein can run at approximately 220 kDa on a non reducing SDS-PAGE and at approximately 195 kDa on a reducing SDS-PAGE.

[00229 In one embodiment, the chimeric polypeptide comprising single chain FVIII is purified (or isolated) from the chimeric polypeptide comprising processed FVIII, and at least about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the FVIII portion of the chimeric polypeptide used herein is single chain FVIII. In another embodiment, at least about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% of the FVIII portion of the chimeric polypeptide is single chain FVIII. In other embodiments, about 1%-about 10%, about 5%-about 15%, about 10%-about 20%, about 15%-about 25%, about 20%-about 30%, about 25%-about 35%, about 30%-about 40% of the FVIII portion of the chimeric polypeptide used herein is single chain FVIII.

[00230] In a particular embodiment, about 1%, about 5%, about 10%, about 15%, about 20% or about 25% of the FVIII portion of the chimeric polypeptide used herein is single chain FVIII. In other embodiments, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the FVIII portion of the chimeric polypeptide used herein is single chain FVIII. In some embodiments, the ratio of the single chain FVIII to the processed FVIII of the chimeric polypeptide is (a) about 25% of single chain FVIII and about 75% of processed FVIII; (b) about 20% of single chain FVIII and about 80% of processed FVIII; (c) about 15% of single chain FVIII and about 85% of processed FVIII; (d) about 10% of single chain FVIII and about 90% of processed FVIII; (e) about 5% of single chain FVIII and about 95% of processed FVIII; (f) about 1% of single chain FVIII and about 99% of processed FVIII; or (g) about 100% of processed FVIII.

[00231J In other embodiments, the ratio of the single chain FVIII to the processed FVIII of the chimeric polypeptide is (a) about 30% of single chain FVIII and about 70% of processed FVIII; (b) about 40% of single chain FVIII and about 60% of processed FVIII; (c) about 50% of single chain FVIII and about 50% of processed FVIII; (d) about 60% of single chain FVIII and about 40% of processed FVIII; (e) about 70% of single chain FVIII and about 30% of processed FVIII; (f) about 80% of single chain FVIII and about 20% of processed FVIII; (g) about 90% of single chain FVIII and about 10% of processed FVIII; (h) about 95% of single chain FVIII and about 5% of processed FVIII; (i) about 99% of single chain FVIII and about 1% of processed FVIII; or (j) about 100% of single chain FVIII.

[00232] The FVIII portion in the chimeric polypeptide used herein has VIII activity. VIII activity can be measured by any known methods in the art. For example, one of those methods can be a chromogenic assay. The chromogenic assay mechanism is based on the principles of the blood coagulation cascade, where activated FVIII accelerates the conversion of Factor X into Factor Xa in the presence of activated Factor IX, phospholipids and calcium ions. The Factor Xa activity is assessed by hydrolysis of a p-nitroanilide (pNA) substrate specific to Factor Xa. The initial rate of release of p-nitroaniline measured at 405 nM is directly proportional to the Factor Xa activity and thus to the FVIII activity in the sample.

[00233] The chromogenic assay is recommended by the FVIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostatsis (ISTH). Since 1994, the chromogenic assay has also been the reference method of the European Pharmacopoeia for the assignment of FVIII concentrate potency. Thus, in one embodiment, the chimeric polypeptide comprising single chain FVIII has FVIII activity comparable to a chimeric polypeptide comprising processed FVIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc portions and processed FVIII, wherein said processed FVIII is fused to one of the two Fc portions), when the FVIII activity is measured in vitro by a chromogenic assay.

100234] In another embodiment, the chimeric polypeptide comprising single chain FVIII of this disclosure has a Factor Xa generation rate comparable to a chimeric polypeptide comprising processed FVIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc portions and processed FVIII, wherein the processed FVIII is fused to one Fc of the two Fc portions).

[00235] In order to activate Factor X to Factor Xa, activated Factor IX (Factor IXa) hydrolyzes one arginine-isoleucine bond in Factor X to form Factor Xa in the presence of Ca 2 , membrane phospholipids, and a FVIII cofactor. Therefore, the interaction of FVIII with Factor IX is critical in coagulation pathway. In certain embodiments, the chimeric polypeptide comprising single chain FVIII can interact with Factor IXa at a rate comparable to a chimeric polypeptide comprising processed FVIII (e-g., a chimeric polypeptide consisting essentially of or consisting of two Fe portions and processed FVIII, wherein the processed FVIII is fused to one Fe of the two Fe portions).

[00236] In addition, FVIII is bound to von Willebrand Factor while inactive in circulation. FVIII degrades rapidly when not bound to vWF and is released from vWF by the action of thrombin. In some embodiments, the chimeric polypeptide comprising single chain FVIII binds to von Willebrand Factor at a level comparable to a chimeric polypeptide comprising processed FVIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fe portions and processed FVIII, wherein the processed FVIII is fused to one Fe of the two Fc portions).

[00237] FVIII can be inactivated by activated protein C in the presence of calcium and phospholipids. Activated protein C cleaves FVIII heavy chain after Arginine 336 in the Al domain, which disrupts a Factor X substrate interaction site, and cleaves after Arginine 562 in the A2 domain, which enhances the dissociation of the A2 domain as well as disrupts an interaction site with the Factor IXa. This cleavage also bisects the A2 domain (43 kDa) and generates A2-N (18 kDa) and A2-C (25 kDa) domains. Thus, activated protein C can catalyze multiple cleavage sites in the heavy chain. In one embodiment, the chimeric polypeptide comprising single chain FVIII is inactivated by activated Protein C at a level comparable to a chimeric polypeptide comprising processed FVIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fe portions and processed FVIII, wherein the processed FVIII is fused to one Fc of the two Fe portions).

[00238] In other embodiments, the chimeric polypeptide comprising single chain FVIII has FVIII activity in vivo comparable to a chimeric polypeptide comprising processed VITI (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fe portions and processed FVIII, wherein the processed FVIII is fused to one Fc of the two Fc portions). In a particular embodiment, the chimeric polypeptide comprising single chain FVIII is capable of protecting a HemA mouse at a level comparable to a chimeric polypeptide comprising processed FVIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc portions and processed FVIII, wherein said processed FVIII is fused to one Fe of the two Fc portions) in a HemA mouse tail vein transection model.

[00239] The term "comparable" as used herein means a compared rate or level resulted from using the chimeric polypeptide is equal to, substantially equal to, or similar to the reference rate or level. The term "similar" as used herein means a compared rate or level has a difference ofno more than 10% or no more than 15% from the reference rate or level (e.g., FXa generation rate by a chimeric polypeptide consisting essentially of or consisting of two Fe portions and processed FVIII, wherein said processed FVIII is fused to one Fe of the two Fe portions). The term "substantially equal" means a compared rate or level has a difference of no more than 0.01%, 0.5% or 1% from the reference rate or level.

[00240] The present disclosure further includes a composition comprising a chimeric polypeptide having FVIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% of the chimeric polypeptide comprises a FVIII portion, which is single chain FVIII and a second portion. In another embodiment, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the chimeric polypeptide in the composition is single chain FVIII. In other embodiments, the second portion is an Fc. In yet other embodiments, the chimeric polypeptide comprises another half-life extending moiety, e.g., albumin.

[00241] In still other embodiments, the composition of the present disclosure comprises a combination of a chimeric polypeptide comprising processed FVIII and a chimeric polypeptide comprising single chain FVIII, (a) wherein about 30% of the FVIII portion of the chimeric polypeptide is single chain FVIII, and about 70% of the FVIII portion of the chimeric polypeptide is processed FVIII; (b) wherein about 40% of the FVIII portion of the chimeric polypeptide is single chain FVIII, and about 60% of the FVIII portion of the chimeric polypeptide is processed FVIII; (c) wherein about 50% of the FVIII portion of the chimeric polypeptide is single chain FVIII, and about 50% of the FVIII portion of the chimeric polypeptide is processed FVIII; (d) wherein about 60% of the FVIII portion of the chimeric polypeptide is single chain FVIII and about 40% of the FVIII portion of the chimeric polypeptide being processed FVIII; (e) wherein about 70% of the FVIII portion of the chimeric polypeptide is single chain FVIII and about 30% of the FVIII portion of the chimeric polypeptide is processed FVIII; (f) wherein about 80% of the FVIII portion of the chimeric polypeptide is single chain FVIII and about 20% of the FVIII portion of the chimeric polypeptide is processed FVIII; (g) wherein about 90% of the FVIII portion of the chimeric polypeptide is single chain FVIII and about 10% of the FVIII portion of the chimeric polypeptide is processed FVIII; (h) wherein about 95% of the FVIII portion of the chimeric polypeptide is single chain FVIII and about 5% of the FVIII portion of the chimeric polypeptide is processed FVIII; (i) wherein about 99% of the FVIII portion of the chimeric polypeptide is single chain FVIII and about 1% of the FVIII portion of the chimeric polypeptide is processed FVIII; or () wherein about 100% of the VIII portion of the chimeric polypeptide is single chain FVIII.

[00242] In certain embodiments, the composition of the present disclosure has FVIII activity comparable to the composition comprising processed FVIII (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fe portions and processed FVIII, wherein said processed FVIII is fused to one of the two Fe portions), when the FVIII activity is measured in vitro by a chromogenic assay.

[00243] In other embodiments, the composition of the disclosure has a Factor Xa generation rate comparable to a composition comprising processed FVIII (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fc portions and processed FVIII, wherein the processed FVIII is fused to one Fe of the two Fc portions). In still other embodiments, the composition comprising single chain FVIII can interact with Factor IXa at a rate comparable to a composition comprising processed FVIII (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fe portions and processed FVIII, wherein the processed FVIII is fused to one Fe).

[00244] In further embodiments, the single chain FVIII in the chimeric polypeptide of the present composition is inactivated by activated Protein C at a level comparable to processed FVIII in a chimeric polypeptide of a composition (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fe portions and processed FVIII, wherein the processed FVIII is fused to one Fe of the two Fc portions). In a particular embodiment, the composition comprising single chain FVIII has FVIII activity in vivo comparable to the composition comprising processed FVIII (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fc portions and processed FVIII, wherein the processed FVIII is fused to one Fe of the two Fc portions). In some embodiments, the composition comprising single chain FVIII of the present disclosure is capable of protecting HemA mouse at a level comparable to the composition comprising processed FVIII (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fe portions and processed FVIII, wherein said processed FVIII is fused to one Fe of the two Fc portions) in HemA mouse tail vein transection model.

[00245] The present disclosure further provides a method for treating a bleeding condition in a human subject using the compositions disclosed herein. An exemplary method comprises administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition/formulation comprising a chimeric polypeptide having FVIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% of the chimeric polypeptide comprises a FVIII portion, which is single chain FVIII, and a second portion. 1002461 The bleeding condition can be caused by a blood coagulation disorder. A blood coagulation disorder can also be referred to as a coagulopathy. In one example, the blood coagulation disorder, which can be treated with a pharmaceutical composition of the current disclosure, is hemophilia or von Willebrand disease (vWD). In another example, the blood coagulation disorder, which can be treated with a pharmaceutical composition of the present disclosure is hemophilia A.

[002471 In some embodiments, the type of bleeding associated with the bleeding condition is selected from hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath.

[00248] In other embodiments, the subject suffering from bleeding condition is in need of treatment for surgery, including, e.g., surgical prophylaxis or peri-operative management. In one example, the surgery is selected from minor surgery and major surgery. Exemplary surgical procedures include tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra abdominal surgery, intrathoracic surgery, joint replacement surgery (e.g., total knee replacement, hip replacement, and the like), heart surgery, and caesarean section.

[00249] In another example, the subject is concomitantly treated with FIX. Because the compounds of the present disclosure are capable of activating FIXa, they could be used to pre-activate the FIXa polypeptide before administration of the FIXa to the subject.

[002501 The methods disclosed herein can be practiced on a subject in need of prophylactic treatment or on-demand treatment.

[00251] The pharmaceutical compositions comprising at least 30% of single chain FVIII can be formulated for any appropriate manner of administration, including, for example, topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration.

[00252] The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. The composition can be also for example a suspension, emulsion, sustained release formulation, cream, gel or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[00253] In one example, the pharmaceutical formulation is a liquid formulation, e.g., a buffered, isotonic, aqueous solution. In another example, the pharmaceutical composition has a pH that is physiologic, or close to physiologic. In other examples, the aqueous formulation has a physiologic or close to physiologic osmolarity and salinity. It can contain sodium chloride and/or sodium acetate. In some examples, the composition of the present disclosure is lyophilized. In some embodiments, the pharmaceutical composition does not comprise an immune cell. In some embodiments, the pharmaceutical composition does not comprise a cell.

[00254] The present disclosure also provides a kit comprising (a) a pharmaceutical composition comprising a chimeric polypeptide which comprises a clotting factor portion and an Fc portion and a pharmaceutically acceptable carrier, and (b) instructions to administer to the composition to a subject in need of immune tolerance to the clotting factor. In some embodiments, the chimeric polypeptide in the kit comprises a FVIII portion, a FVII portion, or a FIX portion. In other embodiments, the chimeric polypeptide in the kit is a FVIII monomer dimer hybrid, a FVII monomer dimer hybrid, or a FIX monomer dimer hybrid. In some embodiments, the instructions further include at least one step to identify a subject in need of immune tolerance to the clotting factor. In some embodiments, the step to identify the subjects in need of immune tolerance includes one or more from the group consisting of: (i) identifying a subject having a mutation or deletion in the clotting factor gene; (ii) identifying a subject having a rearrangement in the clotting factor gene; (iii) identifying a subject having a relative who has previously developed an inhibitory immune response against the clotting factor; (iv) identifying a subject receiving interferon therapy; (v) identifying a subject receiving anti-viral therapy; (vi) identifying a subject having a genetic mutation in a gene other than the gene encoding the clotting factor which is linked with an increased risk of developing an inhibitory immune response; and (vii) two or more combinations thereof.

[002551 In some embodiments, the genetic mutation in a gene other than the gene encoding the clotting factor comprises one or more mutations selected from the group consisting of: (i) a genetic polymorphism associated with increased TNF-a; (ii) a genetic polymorphism associated with increased IL10; (iii) a genetic polymorphism associated with decreased CTLA-4; (iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and (v) has two or more combinations thereof.

[00256] Optionally associated with the kit's container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

EMBODIMENTS

[00257] El. A method of inducing immune tolerance to a clotting factor in a subject in need thereof comprising administering to the subject a chimeric polypeptide, wherein the chimeric polypeptide comprises a clotting factor portion and an Fe portion.

[002581 E2. A method of preventing or inhibiting development of an inhibitor to a clotting factor comprising administering to a subject in need of immune tolerance to the clotting factor a chimeric polypeptide, wherein the chimeric polypeptide comprises a clotting factor portion and an Fe portion.

[002591 E3. The method of embodiment El or E2, wherein the subject would develop an inhibitory immune response against the clotting factor if administered an equivalent dose of a polypeptide consisting of the clotting factor.

[00260] E4. The method of any one of embodiments El to E3, wherein the subject has developed an inhibitory immune response against the clotting factor.

[00261] E5. The method of any one of embodiments El to E4, wherein the subject has never been previously treated with the clotting factor.

[00262] E6. The method of any one of embodiments El to E5, wherein the clotting factor portion comprises Factor VIII, Factor IX, Factor VII, Von Willebrand Factor, or a fragment thereof.

[00263] E7. The method of any one of embodiments El to E5, where in the subject is a fetus and the method further comprises administering the chimeric polypeptide to the mother of the fetus and the administration to the subject occurs from the mother across the placenta.

[00264] E8. The method of embodiment E7, wherein the clotting factor moiety comprises Factor VIII, Factor IX, Factor VII, Von Willebrand Factor, or a fragment thereof.

[00265 E9. The method of any one of embodiments El to E6, wherein the subject is a child or an adult.

[00266] E10. The method of embodiment E9, wherein the subject is a child less than one-year old, less than two-year-old, less than three-year-old, less than four-year-old, less than five year-old, less than six-year-old, less than seven-year-old, less than eight-year-old, less than nine-year-old, less than ten-year-old, less than eleven-year-old, or less than twelve-year-old.

1002671 E11. The method of embodiment E10, wherein the child is less than one-year old.

[00268] E12. The method of embodiment El1, wherein the child or adult develops a bleeding disorder, wherein the onset of the symptoms of the bleeding disorder is after the one-year-old age.

[00269] E l3. The method of any one of embodiments El to E12 wherein the administration is sufficient to prevent, inhibit, or reduce development of an immune response selected from a humoral immune response, a cell-mediated immune response, or both a humoral immune response and a cell-mediated immune response against the clotting factor.

[00270] E14. The method of any one of embodiments El to E13, wherein the composition is administered in repeated doses.

[00271] E15. The method of embodiment E14, wherein each of the repeated doses is separated from another by at least about 12 hours, at least about 24 hours, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, at least about ten days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days. 100272] E16. The method of embodiment E14 or E15, wherein the repeated doses comprise at least about two doses, at least about five doses, at least about 10 doses, at least about 20 doses, at least about 25 doses, at least about 30 doses, at least about 35 doses, at least about 40 doses, at least about 45 doses, at least about 50 doses, at least about 55 doses, at least about 60 doses, at least about 65 doses, or at least about 70 doses.

[00273] E17. The method of embodiment E16, wherein the repeated doses comprise from about two doses to about 100 doses, from about five doses to about 80 doses, from about 10 doses to about 70 doses, from about 10 doses to about 60 doses, from about 10 doses to about 50 doses, from about 15 doses to about 40 doses, from about 15 doses to about 30 doses, from about 20 doses to about 30 doses, or from about 20 doses to about 40 doses.

[002741 E18. The method of embodiment E16 or E17, wherein the repeated doses comprise about two doses, about five doses, about 10 doses, about 15 doses, about 20 doses, about 25 doses, about 30 doses, about 35 doses, about 40 doses, about 45 doses, about 50 doses, about 55 doses, about 60 doses, about 65 does, about 70 doses, about 75 doses, about 80 doses, about 90 doses, or about 100 doses.

[002751 E19. The method of any one of embodiments E14 to E18, wherein the subject is further administered, after the repeated doses, a pharmaceutical composition comprising a clotting factor protein which comprises the clotting factor, but does not comprise an Fc portion. 1002761 E20. The method of embodiment E19, wherein the clotting factor protein is full length or mature clotting factor.

[00277] E21. The method of embodiment E19, wherein the clotting factor protein comprises one or more half-life extending moiety other than an Fc portion.

[002781 E22. The method of any one of embodiments El to E21, wherein the administration treats one or more bleeding episodes.

[00279] E23. The method of any one of embodiments El to E22, wherein the administration prevents one or more bleeding episodes.

[00280] E24. The method of any one of embodiments E l to E23, wherein the administration is an episodic treatment of one or more bleeding episodes.

[00281] E25. The method of any one of embodiments El to E23, wherein the subject is in need of surgical prophylaxis, peri-operative management, or treatment for surgery.

[00282] E26. The method of embodiment E25, wherein the surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra abdominal surgery, intrathoracic surgery, or joint replacement surgery.

[00283] E27. The method of any one of embodiments E13 to E26, wherein the immune response comprises production of inhibitory antibodies to the clotting factor.

[002841 E28. The method of embodiment E27, wherein the antibody concentration is at least 0.6 Bethesda Units (BU).

[00285] E29. The method of embodiment E28, wherein the antibody concentration is at least 5 BU.

[00286] E30. The method of any one of embodiments E13 to E26, wherein the immune response comprises a cell-mediated immune response.

[00287] E31. The method of embodiment E30, wherein the cell-mediated immune response comprises the release of a cytokine selected from the group consisting of IL-12, IL-4, and TNF-a.

[00288] E32. The method of any one of embodiments E13 to E31, wherein the immune response comprises a clinical symptom selected from the group consisting of: increased bleeding tendency, high clotting factor consumption, lack of response to clotting factor therapy, decreased efficacy of clotting factor therapy, and shortened half-life of clotting factor.

[00289] E33. The method of any one of embodiments El to E32, wherein the subject has a mutation or deletion in the clotting factor gene.

[00290] E34. The method of any one of embodiments El to E32, wherein the subject has a rearrangement in the clotting factor gene.

[002911 E35. The method of any one of embodiments El to E34, wherein the subject has severe hemophilia.

[00292] E36. The method of any one of embodiments El to E34, wherein the subject has a relative who has previously developed an inhibitory immune response against the clotting factor.

[00293] E37. The method of any one of embodiments El to E36, wherein the subject is receiving interferon therapy.

[00294] E38. The method of any one of embodiments El to E37, wherein the subject is receiving anti-viral therapy.

[00295] E39. The method of any one of embodiments El to E38, wherein the subject has a genetic polymorphism associated with increased TNF-a.

[002961 E40. The method of embodiment E39, wherein the polymorphism is TNF-a 308G>A.

[00297 E41. The method of any one of embodiments El to E40, wherein the subject has a genetic polymorphism associated with increased IL10.

[002981 E42. The method of embodiment E41, wherein the polymorphism is allele 134 of the ILlOG microsatellite.

[002991 E43. The method of any one of embodiments El to E42, wherein the subject has a genetic polymorphism associated with decreased CTLA-4 expression.

[00300] E44. The method of any one of embodiments E Ito E43, wherein the subject has a mutation in DR15 or DQB0602 MHC Class II molecules.

[003011 E45. The method of any one of embodiments El to E44, wherein the subject has had less than 150 clotting factor exposure days (ED).

[00302 E46. The method of embodiment E45, wherein the subject has had less than 50 ED.

[00303J E47. The method of embodiment E46, wherein the subject has had less than 20 ED. 1003041 E48. The method of any one of embodiment E3 to E47, wherein the inhibitory FVIII immune response developed in response to a full length or mature FVIII clotting factor.

[003051 E49. The method of embodiment E3-E48, wherein the inhibitory immune response is an inhibitory FVIII response developed in response to a recombinant FVIII product.

[00306] E50. The method of any one of embodiments El to E49, wherein the administration reduces the number of antibodies to FVIII in the subject compared to the number prior to administration.

[00307] E51. The method of any one of embodiments El to E50, wherein the administration reduces the titer of antibodies to FVIII in the subject compared to the titer prior to administration.

[003081 E52. The method of any one of embodiments El to E51, wherein the administration reduces the level of a cytokine in the subject compared to the level prior to administration.

[00309] E53. The method of any one of embodiments El to E52, wherein the administration reduces the number of antibodies to FVIII in the subject compared to the number in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

[003101 E54. The method of any one of embodiments El to E53, wherein the administration reduces the titer of antibodies to FVIII in the subject compared to the titer in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

1003111 E55. The method of any one of embodiments El to E54, wherein the administration reduces the level of a cytokine in the subject compared to the level in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

[00312] E56. The method of any one of embodiments El to E55, wherein the administration reduces the number of anti-clotting factor antibodies in the subject compared to the number that would result from administration of polypeptide consisting of the clotting factor portion to the subject.

[003131 E57. The method of any one of embodiments El to E56, wherein the administration reduces the titer of anti-clotting factor antibodies in the subject compared to the titer that would result from administration of polypeptide consisting of the clotting factor portion to the subject. 1003141 E58. The method of any one of embodiments El to E57, wherein the administration reduces the level of a cytokine in the subject compared to the level that would result from administration of polypeptide consisting of the clotting factor portion to the subject.

[00315] E59. The method of embodiment E52, E55, or E58, wherein the cytokine is selected from the group consisting of IL-12, IL-4, and TNF-a.

1003161 E60. The method of any one of embodiments El to E59, which further comprises prior to administration of the chimeric polypeptide, identifying that the subject has one or more characteristics selected from the group consisting of: (a) has a mutation or deletion in the gene encoding the clotting factor; (b) has a rearrangement in the gene encoding the clotting factor; (c) has a relative who has previously developed an inhibitory immune response against the clotting factor; (d) is receiving interferon therapy; (e) is receiving anti-viral therapy; (f) has a genetic mutation in a gene other than the gene encoding the clotting factor which is linked with an increased risk of developing an inhibitory immune response; and (g) two or more combinations thereof.

[00317] E61. The method of embodiment E60, wherein the genetic mutation in a gene other than the gene encoding the clotting factor comprises one or more mutations selected from the group consisting of: (i) a genetic polymorphism associated with increased TNF-a; (ii) a genetic polymorphism associated with increased IL10; (iii) a genetic polymorphism associated with decreased CTLA-4; (iv) a mutation in DR15 or DQB0602 MIC Class II molecules; and (v) has two or more combinations thereof.

[00318] E62. The method of embodiment E61, wherein the polymorphism associated with increased TNF-a is 308G>A.

[00319] E63. The method of embodiment E61, wherein the polymorphism associated with increased IL10 is allele 134 of the IL1OG microsatellite.

[00320] E64. The method of any one of embodiments E6, and E8 to E62, wherein the FVIII portion comprises the FVIII A3 domain.

[00321] E65. The method of any one of embodiments E6, and E8 to 63, wherein the FVIII portion comprises human FVII.

[00322] E66. The method of any one of embodiments E6, and E8 to E64, wherein the FVIII portion has a fill or partial deletion of the B domain. 1003231 E67. The method of any one of embodiments E6, and E8 to E66, wherein the FVIII portion is at least 90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6).

[00324] E68. The method of any one of embodiments E6, and E8 to E66, wherein the FVIII portion is identical to a FVIII amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 20 to 2351 of SEQ ID NO:6).

[00325] E69. The method of any one of embodiments E6, and E8 to E66, wherein the FVIII portion is at least 90% or 95% identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6).

[00326] E70. The method of any one of embodiments E6, and E8 to E66, wherein the FVIII portion is identical to a FVIII amino acid sequence shown in TABLE 2 with a signal sequence (amino acids 1to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6).

[00327] E71. The method of any one of embodiments E6, and E8 to E70, wherein the FVIII portion has coagulation activity.

[00328] E72. The method of any one of embodiments El to E71, wherein the Fc portion is identical to the Fe amino acid sequence shown in TABLE 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6).

[00329] E73. The method of any one of embodiments El to E72, wherein the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide in association with the chimeric polypeptide, wherein the second polypeptide consists essentially of or consists of the Fc portion or the FcRn binding partner. 100330J E74. The method of any one of embodiments El to E73, wherein the chimeric polypeptide is administered at each dose of 10-100 IU/kg.

[003311 E75. The method of embodiment E74, wherein the dose is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg.

[003321 E76. The method of embodiment E75, wherein the dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg. 100333] E77. The method of any one of embodiments El to E76, wherein the subject has a bleeding condition selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath.

[00334] E78. The method of embodiment E77, wherein the bleeding coagulation disorder is hemophilia A.

[00335] E79. The method of any one of embodiments E6, and E8 to E63, wherein the clotting factor portion comprises Factor IX.

[00336] E80. The method of embodiment E79, wherein the Factor IX portion is at least 90%, 95%, or 100% identical to a FIX amino acid sequence shown in TABLE 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID NO:2; amino acids 20 to 2351 of SEQ ID NO:6).

[00337] E81. The method of any one of embodiments E6, and E8 to E63, wherein the chimeric polypeptide is a monomer dimer hybrid comprising a first chain comprising a FIX portion and the Fc portion or the FcRn binding partner and a second chain consisting essentially of or consisting of a Fe portion.

[00338] E82. The method of any one of embodiments E6, and E8 to E63, wherein the chimeric polypeptide comprises a Factor VII portion.

[00339] E83. The method of embodiment E82, wherein the chimeric polypeptide is a monomer dimer hybrid comprising a first chain comprising a FVII portion and the Fc portion and a second chain consisting essentially of or consisting of a Fe portion.

[00340] E84. The method of embodiment E82 or E83, wherein the FVII portion is inactive FVII, activated FVII, or activatable FVII.

[003411 E85. A kit comprising (a) a pharmaceutical composition comprising a chimeric polypeptide which comprises a clotting factor portion and an Fc portion or an FcRn binding partner portion and a pharmaceutically acceptable carrier, and (b) instructions to administer to the composition to a subject in need of immune tolerance to the clotting factor.

[003421 E86. The kit of embodiment E85, wherein the chimeric polypeptide comprises a FVIII portion, a FVII portion, or a FIX portion.

[00343] E87. The kit of embodiment E86, wherein the chimeric polypeptide is a FVIII monomer dimer hybrid, a FVII monomer dimer hybrid, or a FIX monomer dimer hybrid.

[003441 E88. The kit of any one of embodiments E85 to E87, wherein the instructions further include at least one step to identify a subject in need of immune tolerance to the clotting factor.

[003451 E89. The kit of embodiment E88, wherein the step to identify the subjects in need of immune tolerance includes one or more from the group consisting of: (a) identifying a subject having a mutation or deletion in the clotting factor gene; (b) identifying a subject having a rearrangement in the clotting factor gene; (c) identifying a subject having a relative who has previously developed an inhibitory immune response against the clotting factor; (d) identifying a subject receiving interferon therapy; (e) identifying a subject receiving anti-viral therapy; (f) identifying a subject having a genetic mutation in a gene other than the gene encoding the clotting factor which is linked with an increased risk of developing an inhibitory immune response; and (g) two or more combinations thereof.

[003461 E90. The kit of claim E89, wherein the genetic mutation in a gene other than the gene encoding the clotting factor comprises one or more mutations selected from the group consisting of: (i) a genetic polymorphism associated with increased TNF-; (ii) a genetic polymorphism associated with increased IL10; (iii) a genetic polymorphism associated with decreased CTLA-4; (iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and (v) has two or more combinations thereof.

[00347] E91. The method of any one of embodiments El to E84, further comprising measuring the level of an inhibitory immune response after the administration.

[003481 E92. The method of embodiment E91, wherein further comparing the level of the inhibitory immune response after the administration with the level of the inhibitory immune response before the administration.

[003491 E93. The method of embodiments E91 or E92, wherein the inhibitory immune response is development of antibodies against FVIII.

[003501 E94. The method of E91 or E92, wherein the inhibitory immune response is cytokine secretion.

[003511 Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. All patents and publications referred to herein are expressly incorporated by reference.

Examples

Example 1 Cloning, expression and purification of rFVIIIFc

[00352] All molecular biology procedures were performed following standard techniques. The coding sequence of human FVIII (Genbank accession number NM_000132), including its native signal sequence, was obtained by reverse transcription-polymerase chain reactions (RT-PCR) from human liver polyA RNA. Due to the large size of FVIII, the coding sequence was obtained in several sections from separate RT-PCR reactions, and assembled through a series of PCR reactions, restriction digests and ligations into an intermediate cloning vector containing a B domain deleted (BDD) FVIII coding region with a fusion of serine 743 (S743) to glutamine 1638 (Q1638), eliminating 2682 bp from the B domain of full length FVIIL The human IgGI Fe sequence (e.g., GenBank accession number Y14735) was obtained by PCR from a leukocyte cDNA library, and the final expression cassette was made in such a way that the BDD FVIII sequence was fused directly to the N-terminus of the Fe sequence (hinge, CH2 and CH3 domains, beginning at D221 of the IgGI sequence, EU numbering) with no intervening linker. For expression of the Fe chain alone, the mouse IgK (kappa) light chain signal sequence was created with synthetic oligonucleotides and added to the Fe coding sequence using PCR to enable secretion of this protein product. The FVIIIFc and Fe chain coding sequences were cloned into a dual expression vector, pBudCE4.1 (Invitrogen, Carlsbad, CA).

[00353] HEK 293H cells (Invitrogen, Carlsbad, CA) were transfected with the pSYN-FVIII 013 plasmid using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA)), and a stable cell line was selected with zeocin. Cells were grown in serum free suspension culture, and rFVIIIFc protein purified from clarified harvest media using a four column purification process, including a FVIII-specific affinity purification step (McCue J. et al, J. Chromatogr. A., 1216(45): 7824-30 (2009)), followed by a combination of anion exchange columns and a hydrophobic interaction column.

Example 2 Characterization of rFVIIIFc (a) Biochemical Characterization

[00354] Processed recombinant FVIII-Fc (rFVIIIFc) was synthesized as two polypeptide chains, one chain consisting of BDD-FVIII (S743-Q1638 fusion, 1438 amino acids) fused to the Fc domain (hinge, CH2 and CH3 domains) of IgG (226 amino acids, extending from D221 to G456, EU numbering), for a total chain length of 1664 amino acids, the other chain consisting of the same Fc region alone (226 amino acids). Though cells transfected with the FVIIIFc/Fe dual expression plasmid were expected to secrete three products (FVIIIFc dimer, FVIIIFc monomer, and Fe dimer), only the FVIIIFc monomer and Fc dimer were detected in conditioned media. Purified FVIIIFc was analyzed by non-reducing and reducing SDS-PAGE analysis (FIG. 2A and 2B). For the nonreduced SDS-PAGE, bands were found migrating at approximately 90 kDa and 130 kDa, consistent with the predicted molecular weights of the FVIIIFc heavy chain (HC) and light chain-dimeric Fe fusion (LCFc2) (FIG. 2A, lane 3). A third band was also detected at approximately 220 kDa, consistent with the predicted molecular weight for single chain FVIIIFc (SC FVIIIFc; HC+LCFc2), in which the arginine residue at position 754 (1648 with respect to the full length sequence) is not cleaved during secretion. For the reduced SDS-PAGE analysis, major bands were seen migrating at approximately 25 kDa, 90 kDa, 105 kDa, and 195 kDa, consistent with the predicted molecular weights for the single chain Fe, HC, LCFc, and SC FVIIIFe (FIG. 2B, lane 3). Cotransfection with human PC5, a member of the proprotein convertase subtlisin/kexin (PCSK) type proteases, resulted in full processing of the rFVIIIFc product (FIG. 2A, 2B, lane 2).

[003551 Densitometry analysis of several batches of rFVIIIFc after SDS-PAGE indicated greater than 98% purity of the expected bands. Size exclusion chromatography (SEC) was also used to assess the degree of aggregation present, and all batches were found to have aggregate levels at 0.5% or less.

[00356] rFVIIIFc structure was further analyzed by thrombin cleavage, reduction, and analysis by LC/UV and LC/MS. The four FVIII fragments generated by thrombin (by cleavages at three arginine residues, at positions 372, 740 and 795 (795 corresponds to 1689 with respect to the full length FVIII sequence), can be detected by UV absorbance (FIG. 2C), corresponding to the following segments of the protein: Fe (peak 1), light-chain-Fe (peak 2); the Al domain from the heavy chain (peak 3) and the A2 domain from the heavy chain (peak 4). The 14 amino acid B domain linker and ~ 6 kDa a3-related peptides are not detected by UV absorbance due to their small size. 100357] The rFVIIIFc polypeptide produced without cotransfected processing enzymes exhibited 15-25% single chain FVIIIFc (SC FVIIIFc), which differs from processed rFVIIIFc by a single peptide bond between R754 and E755 (R1648/El649 with respect to the full length FVIII). This isoform was purified and characterized in all of the biochemical assays described above, and found to be comparable to rFVIIIFc as shown below. The activity of purified single chain FVIIIFc was found to be similar to rFVIIIFc in a chromogenic assay as well as by the various functional assays described below.

(b) Measurement of FVIIIActivity by Chromogenic and One-Stage aPTTAssays

[00358] FVIII activity was measured by a FVIII chromogenic assay. The average specific activity from four separate batches of rFVIIIFc was found to be 9762 ±449 IU/mg by the chromogenic assay, corresponding to 214899 IU/nmol. FVIII activity of single chain FVIII:Fc was also measured by the chromogenic assay and compared to the completely processed rFVIIIFc or rFVIIIFc DS (containing about 25% single chain rFVIIIFc). As TABLE 3A shows, single chain rFVIIIFc showed no significant difference in FVIII activity compared to the FVIII activity of completely processed FVIIIFc or rFVIIIFc DS by the chromogenic assay, both in the presence and the absence of von Willebrand Factor (VWF). TABLE 3B shows that full activity of SCrFVIIIFc, as measured by one-stage activated partial thromboplastin time (aPTT) assay, was observed in the absence of VWF.

TABLE 3A: FVIII Activity by Chromogenic Assay

Matrix Sample Chromogenic %CV* Specific Activity(IU/mg) rFVIIIFcDS (25% NP) (RECD-19189- 9066 2.49 09-013) FVIII depleted Single chain rFVIIIFc (purified from 8194 2.72 plasma RECD 19189-09-013) Completely Processed rFVIIIFc 9577 8.34 (purified from an engineered cell line) rFVIIIFcDS (25% NP) (RECD-19189- 10801 8.92 FVIII and 09-013) vWFdepleted Single chain rFVIIIFc 9498 4.70 plasma (purified from RECD 19189-09-013)

Completely Processed rFVIIIFc 9569 4.54 (purified from an engineered cell line) *CVzcoefficient of variation

TABLE 3B: FVIII Activity by aPTT assay

Matrix Sample Coagulation %CV (aPTT) Specific Activity (IU/mg) rFVIIFcDS (25% NP) 8210 5.88 (RECD-19189-09-013) eVIII- Single chain rFVIIIFc 3108 6.57 plasma (purified from RECD 19189-09-013) Completely Processed rFVIIIFc 8683 3.57 (purified from an engineered cell line) rFVIIIFcDS (25% NP) 15621 6.47 FVIII and (RECD-19189-09-013) vWF Single chain rFVIIIFc 13572 2.41 plasma (purified from RECD 19189-09-013) Completely Processed rFVIIIFc 15170 10.42 (purified from an engineered cell line)

(c) Activity in Xase Complex

[00359] FVIII activity was also measured in the context of the Xase complex, by incubating activated FIX and thrombin-activated REFACTO@ or rFVLIIFc protein on a phospholipid surface in the presence of calcium, and monitoring the conversion of FX to FXa as measured by cleavage of a chromogenic or fluorogenic substrate, from which FXa generation rates were determined. This assay was then modified by varying one component of the assay while keeping the others constant in order to examine the interactions with each individual component.

[00360j The FXa generation rate was determined as a function of varying phospholipid concentrations for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFc (FIG. 3A), using synthetic phospholipid vesicles (25% phosphotadyl serine/75% phosphotadyl choline). Both proteins were found to have a similar activity profile, with peak activity at approximately 156 pM phospholipids.

[00361] The FXa generation rate was then determined as a function of varying FX concentrations, and Km and Vma values calculated (FIG. 3B). The activity profiles for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFc were found to be similar, with similar Km and Vm, (TABLE 4). Finally, the FXa generation rate was determined as a function of varying FIX concentrations (FIG. 3C). The activity profiles appeared similar, with similar Kd and V ,8 (TABLE 5). Similar results were obtained using platelets as a phospholipid source (unpublished data, June 2009).

TABLE 4. FXa Generation Parameters for FVIII Proteins on Phospholipids Lipid Source Molecule Km (nM) Vmax (nM/min) rFVIIIFc DS 55.0 ± 5.9 65.6 + 8.6 25%PS-75%PC rBDD FVIII 51.0 + 8.7 73.5 + 10.1 NP rFVIIIFc 53.2 7.5 56.0 13.8

TABLE 5. FIXa Interactions with FVIII Proteins Lipid Source Molecule Km (nM) Vmax (nM/min) rFVIIIFc DS 2.8 ± 0.4 4.5 ± 0.3 25%PS-75%PC rBDD FVIII 2.5 0.3 4.0 + 1.0 NP rFVIIIFc 2.3 + 0.2 3.8 ±z0.4

(d) Inactivation by APC

[00362] Once active, FVIIl is inactivated by cleavage by activated protein C (APC), as well as by dissociation of the A2 domain. rFVIIIFe and rBDD FVIII were both activated by thrombin, then incubated with APC for different times and activity determined in a FXa generation assay (FIG. 4). In the absence of thrombin activation, little FXa generation was detected, and this was increased significantly with thrombin digestion. Treatment with APC for 90 minute led to a significant decrease in FXa generation rates, similar to non-activated samples, and these results were similar for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFc.

(e) Affinityfor vWF

[00363] FVIII interactions with von Willebrand factor (vWF) were measured by real-time biomolecular interaction analysis (BIAcore), based on surface Plasmon resonance (SPR) technology, to determine the kinetics of binding of rFVIIIFc and rBDD FVIII towards vWF (TABLE 6). Kinetic rate parameters of Ka (on-rate) and K (off-rate), and the affinity KD (Kd/Ka), were determined for each FVIII interaction under identical conditions. Both rFVIIIFc and rBDD FVIII were found to have a low nM binding affinity (KD) for vWF, of 1.64±0.37 and 0.846+0.181 nM, respectively. The proteins had similar off-rates, with a two fold difference in on-rate resulting in a two fold difference in the affinity.

TABLE 6. Biocore Binding Analysis of FVIII Proteins to vWF Kinetic rate parameters Off-rate/On-rate Analyte Ligand N On-rate(M-is-i) Off-rate(s-1) KD(M) rFVIIIFc DS hvWf 5 7.92±1.51x105 1.25 1.12x10- 1.64 0.37x10-9 NPrFVIIIFc hvWf 5 8.66 ±1.1Ox10 1.09+0.09x10-3 1.28 0.22x10-9 rBDD FVIII hvWf 5 13.7±1.50xl05 1.14 0.12x10 0.846 ±0.181x10-9

[00364] As shown in TABLE 6, the affinity of rFVIIIFc DS or single chain rFVIIIFc with vWF was found to be in the low nM range, approximately two fold greater than that of BDD FVIII alone. At physiological concentrations, this would result in a slight decrease in the percentage of rFVIlIFc (processed or single chain) complexed with vWF as compared to free FVIII, however in vivo studies have indicated that the half-life of rFVIIIFc is significantly prolonged over full length or BDD FVIII despite this slightly lower affinity, and therefore this does not appear to compromise the half-life of the molecule. The free rFVIIIFc may be more efficiently recycled through the FcRn pathway and therefore contribute to a greater prolongation of half-life.

Example 3 rFVIIIFc Phase I/Ila Clinical Trial

100365] A Phase I/Ila, open-label, crossover, dose-escalation, multi-center, and first-in-human study was designed to evaluate the safety, tolerability, and pharmacokinetics of a single dose of rFVIIIFc in subjects with severe (defined as <1 IU/dL [1%] endogenous FVIII [FVIII]) hemophilia A. A total of approximately 12 previously treated patients were enrolled and dosed with rFVIIIFc at 25 or 65 IU/kg. After the screening (scheduled within 28 days prior to the first dose of the ADVATE* [rFVIII], the reference comparator agent) and a minimum of 4-days (96 hours) elapsing with no FVIII treatment prior to the first injection, approximately 6 subjects received a single 25 IU/kg dose of ADVATE* followed by a 3-day (72 hours) pharnacokinetic (PK) profile then crossover and receive a 25 IU/kg single, open-label dose of rFVIIIFe for a 7-day (168 hours) PK profiling. The first 3 subjects were dosed sequentially. For the first three (3) subjects dosed with 25 IU/kg of rFVIIIFc, each subject underwent an inhibitor assessment at 14-days (336 hours) post-injection of rFVIIIFc. Dosing of the next subject (for the first three subjects only) occurred once the inhibitor testing is completed. After the 3rd subject completed the 14 day inhibitor assessment, the remaining three subjects at 25 IU/kg and the six subjects at 65 IU/kg began enrollment sequentially at least 1 day apart within each dose group.

[00366] One week after the last subject received the 25 IU/kg dose of the rFVIIIFc, approximately 6 unique subjects were recruited for the 65 IU/kg cohort. Each subject in the 65 IU/kg cohort received a single 65 IU/kg dose of ADVATE* followed by a 4-day (96 hours) PK profiling then crossover and receive a 65 IU/kg single, open-label dose of rFVIIIFc for a 10-day (240 hours) profiling. If a bleeding episode occurred before the first injection of rFVIIIFc in any cohort, subject's pre-study FVIII product was used for treatment and an interval of at least 4 days had to pass before receiving the first injection of rFVIIIFc for the PK profile.

[003671 All subjects were followed for a 14-day (336 hours) and 28 day safety evaluation period after administration of rFVIIIFe25 U/kg or 65 IU/kg for safety. All subjects underwent pharmacokinetic sampling pre- and post-dosing along with blood samples for analysis of FVIII activity at designated time points.

[00368] The pharmacokinetic data for the Phase I/Ila clinical trial demonstrated the following results for FVIIIFc. FVIIIFc had about a 50% increase in systemic exposure (AUCINF), about 50% reduction in clearance (Cl), and about 50-70% increase in elimination half-life and MRT compared to ADVATE* (full length rFVIII). In addition, FVIIIFe showed increased C168, TBLP1, TBLP3, and TBLP5 values compared to ADVATEF.

[003691 The measured PK parameters were: AUCNF Area under the concentration-time curve from zero to infinity Beta HL Elimination phase half-life; also referred to as tu2 C168 Estimated FVIIIFc activity above baseline at approximately 168 h after dose

Cl Clearance MRT Mean residence time TBLPI Model-predicted time after dose when FVIIIFc activity has declined to approximately 1 IU/dL above baseline TBLP3 Model-predicted time after dose when FVIIIFc activity has declined to approximately 3 IU/dL above baseline TBLP5 Model-predicted time after dose when FVIIIFc activity has declined to approximately 5 IU/dL above baseline

Example 4 Pharmacokinetics (PK) of rFVIIFe

[003701 A recombinant B-domain-deleted FVIII-Fc (rFVIIIFc) fusion protein has been created as an approach to extend the half-life of FVIII. The pharmacokinetics (PK) of rFVIIIFc were compared to rFVIII in hemophilia A mice. The terminal half-life was twice as long for rFVIIIFc compared to rFVIII. In order to confirm that the underlying mechanism for the extension of half-life was due to the protection of rFVIIIFc by FeRn, the PK were evaluated in FeRn knockout and human FcRn transgenic mice.

[00371] A single intravenous dose (125 IU/kg) was administered and the plasma concentration measured using a chromogenic activity assay. The Cma was similar between rFVIIIFc and rFVIII (XYNTHA@) in both mouse strains. However, while the half-life for rFVIIIFc was comparable to that of rFVIII in the FeRn knockout mice, the half-life for rFVIIIFc was extended to approximately twice longer than that for rFVIII in the hFRn transgenic mice. These results confirmed that FcRn mediated or was responsible for the prolonged half-life of rFVIIIFc compared to rFVIIL Since hemostasis in whole blood measured by rotation thromboelastometry (ROTEM*) has been shown to correlate with the efficacy of coagulation factors in bleeding models of hemophilia mice as well as in clinical applications, we sought to evaluate the ex vivo efficacy of rFVIIIFc in the hemophilia A mice using ROTEM*. 100372] Hemophilia A mice were administered a single intravenous dose of 50 IU/kg rFVIIIFc, XYNTHA* (FVIII) or ADVATE* (FVIII). At 5 minutes post dose, clot formation was similar with respect to clotting time (CT), clot formation time (CFT) and a-angle. However, rFVIIIFc showed significantly improved CT at 72 and 96 hr post dose, and CFT and a-angle were also improved at 96 hours compared to both XYNTHA® (FVIII) and ADVATE* (FVIII), consistent with prolonged PK of rFVIIIFc. These results indicated that rFVIIIFC has a defined mechanism of action resulting in an increased half-life, and the potential to provide prolonged protection from bleeding.

Example 5 rFVIIIFc Phase I/Ila Clinical Trial Results

[00373] This Example presents final analysis results for FVIII activity from 16 patients treated with 25 and 65 IU/kg FVIII products. See Example 3. rFVIIIFc is a recombinant fusion protein comprised of a single molecule of recombinant B-domain deleted human FVIII (BDD-rFVIII) fused to the dimeric Fe domain of the human IgG1, with no intervening linker sequence. This protein construct is also referred to herein as rFVIIIFc heterodimeric hybrid protein, FVIIIFc monomeric Fe fusion protein, FVIIIFc monomer hybrid, monomeric FVIIIFc hybrid, and FVIIIFc monomer-dimer. See Example 1, FIG. 1, and TABLE 2A.

[003741 Preclinical studies with rFVIIIFc showed an approximately 2-fold prolongation of the half-life of rFVIII activity compared to commercially available rFVIII products. The rationale for this study was to evaluate the safety and tolerability of a single dose of rFVIIIFc in frozen liquid formulation and provide data on the PK in severe hemophilia A subjects. For this study, 16 evaluable subjects were available for PK evaluation. Single administration of two doses of both rFVIIIFc and ADVATE* at a nominal dose of 25 (n=6) and 65 IU/kg of body weight (n=10) were infused intravenously over approximately 10 minutes. Blood samples for plasma PK assessments were obtained before infusion, as well as up to 10 days after dosing. The PK of FVIII activity for both ADVATE*and rFVIIIFc were characterized in this study using a model-dependent method.

Objectives

[00375] The primary objective of this study was to assess the safety and tolerability of single administration of two doses of rFVIIIFc (25 and 65 IU/kg) in previously treated patients (PTPs) aged 12 and above with severe hemophilia A. The secondary objective was to determine the pharmacokinetics (PK) parameters determined by pharmacodynamic (PD) activity of FVIII over time after a single administration of 25 or 65 IU/kg of rFVIIIFc compared to ADVATE in one-stage clotting and chromogenic assays.

Study Design (see Example 3)

[00376] Blood samples were collected for FVIII activity PK evaluations at the screening visit (within 28 days prior to dosing ADVATE*); on Day 0 (injection of ADVATE) pre-injection and at 10 and 30 minutes and 1, 3, 6, and 9 hours post-injection; on Day 1 at 24 hours post injection of ADVATE; on Day 2 at 48 hours post-injection of ADVATE*; on Day 3 at 72 hours post-injection of ADVATE*; and on Day 4 at 96 hours post-injection of high dose of ADVATE* (Cohort B only).

[00377] Blood samples were collected for FVIII activity PK evaluations on the day of rFVIIIFc injection just prior to the administration of rFVIIIFc, at 10 and 30 minutes and 1, 3, 6, and 9 hours post-injection of rFVIIIFc; on Day I at 24 hours post-injection of rFVIIIFe; on Days 2 through 5 at 48, 72, 96, and 120 hours post-injection of rFVIIIFc; on Day 7 at 168 hours post-injection of rFVIIIFc; on Days 8, 9, and 10 at 192, 216, and 240 hours post injection of high dose of rFVIIIFc (Cohort B only). FVIII activity was also measured at the final study visit (28 days post-injection of rFVIIIFc) at 672 hours post-injection of rFVIIIFc.

Pharmacokinetic Modeling Abbreviations TBLP1 Model-predicted time after dose when FVIII activity has declined to approximately 1IU/dL above baseline. TBLP3 Model-predicted time after dose when FVIII activity has declined to approximately 3 IU/dL above baseline Calculations KVM = Cm._M/Actual Dose (IU/kg) KV_OB Cma_OB/Actual Dose (IU/kg) IVRM= 100 x Cma_M x Plasma Volume (dL) / Total Dose in IU; where plasma volume in mL = (23.7 x Ht in em) + (9.0 x Wt in kg) - 1709. IVR_OB = 100 x CmxOB x Plasma Volume (dL) / Total Dose in IU; where plasma volume in mL = (23.7 x Ht in cm)+ (9.0 x Wt in kg) - 1709.

Results (a) Single-Dose Pharmacokinetics (One-Stage Assay)

[003781 Observed FVIII activity increased sharply after the short IV infusion of either ADVATE"tor rFVIIIFc, with mean (+SD) model-predicted Cm. values of 56.6 4.74 and 121 28.2 IU/dL for ADVATE* and 55.6 8.18 and 108 + 16.9 IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. All ADVATE- and rFVIIIFe-treated patients had dose-related increases in FVIII activity. The observed increase in both Cm and AUCINF was slightly less than proportional to dose over the dose range evaluated.

[00379] After the end of the infusion, the decline of the observed FVIII activity exhibited monoexponential decay characteristics until the baseline level was reached. The rate of decline in FVIII activity was slower for rFVIIIFc than for ADVATE* with mean (±SD) model-predicted elimination half-life values of 11.9 ±2.98 and 10.4 ±3.03 hr for ADVATE* and 18.0± 3.88 and 18.4 + 6.99 hr for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. Elimination half-life values appeared to be dose-independent over the dose range evaluated for both FVIII products.

[00380] Total systemic FVIII exposure (assessed by AUCINF) was - 48% and 61% greater following rFVIIIFc administration than ADVATE at 25 and 65 IU/kg dose levels, respectively. Mean (SD) model-predicted AUCNF values were 974 i 259 and 1810 ±606 hr*IU/dL for ADVATE* and 1440 316 and 2910 1320 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

[003811 Similar to elimination half-life, the MRT was prolonged for rFVIIIFc relative to ADVATE. Mean (+SD) model-predicted MRT values were 17.1 ±4.29 and 14.9 ±4.38 hr for ADVATEand 25.9 ±5.60 and 26.5 ± 10.1 hr for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRT values appeared to be dose-independent over the dose range evaluated for both FVIII products.

[00382] In addition, primary PK parameter values for CL and V were determined. CL values for rFVIIIFc only accounted for - 66% of those observed for ADVATE at equivalent doses. Mean (±SD) model-predicted CL values were 2.70 + 0.729 and 4.08 1.69 mL/hr/kg for ADVATE" and 1.80 0.409 and 2.69 ±1.25 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. V values were comparable between ADVATE" and rFVIIIFc with mean (+SD) model-predicted V values of 43.9 4.27 and 56.1 13.4 mL/kg for ADVATE® and 45.3 7.23 and 61.6 + 10.6 mL/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. Slight increases in mean CL and V values were noted with increasing dose of ADVATE* and rFVIIIFc; however, the increase in standard deviations at the 65lU/kg dose coupled with limited dose levels confounded an assessment of the dose-dependency of these parameters. For example, the CV% geometric mean CL value for the rFVIIIFc treatment group increased from 23.0% (25 IU/kg) to 48.6% (65 IU/kg).

1003831 In addition to the primary PK parameters, secondary PK parameters (eg. K-values, IVR, etc.) were determined to evaluate FVIII duration of effect. Evidence of PK difference was also observed with rFVIIIFc demonstrating increased TBLPland TBLP3 values compared to ADVATE* at equivalent doses. IVR and K-values for ADVATE* and rFVIIIFc appeared to be comparable. A slight increase in TBLP1 and TBLP3 values were observed with increasing dose of ADVATE and rFVIIIF. In contrast, slight decreases in mean IVR and K-values were noted with increasing dose of ADVATE*and rFVIIIFc. As previously indicated, an assessment of the dose dependency of these parameters is confounded by limited dose levels.

[00384] Mean (SD) observed TBLP1were 2.88 ±0.733 and 2.93 0.848 IU/dL per lU/kg for ADVATE* and 4.28 0.873 and 5.16 2.02 IU/dL per IU/kg for rFVIIIFc for the 25 and 65 lU/kg dose groups, respectively. Mean (+SD) observed TBLP3 were 2.06 0.527 and 2.26 0.666 IU/dL per IU/kg for ADVATE* and 3.09 0.623 and 3.93 1.59 IU/dL per IU/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

[003851 Mean IVR and K-values calculated using observed Cm. values (subtracted with baseline and residual drug within the model) were generally greater than values determined using model-predicted Cma values; consistent with slight underestimation of the observed peak activity using the one-compartment model. Mean (+SD) observed K-values were 2.57± 0.198 and 2.13 0.598 IU/dL per IU/kg for ADVATE and 2.46 ±0.330 and 1.85 ±0.332 IU/dL per IU/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. Mean (+SD) observed IVR values were 94.1 15.6 and 85.8 16.5 % for ADVATE* and 89.5 11.9 and 74.8 ±6.72 % for rFVIIIFe for the 25 and 65lU/kg dose groups, respectively.

(b) Single-Dose Pharmacokinetics (Chromogenic Assay)

1003861 Observed FVIII activity increased sharply after the short IV infusion of either ADVATE* or rFVIIIF, with mean (+SD) model-predicted Cm values of 70.2 ±9.60 and 157 ±38.6 IU/dL for ADVATE* and 70.3 10.0 and 158 34.7 IU/dL for rFVIIIFe for the 25 and 65 IU/kg dose groups, respectively.

[00387] All ADVATE* and rFVIIIFc-treated patients had dose-related increases in FVIII activity. The observed increase in both Cm and AUCINF was slightly less than proportional to dose over the dose range evaluated.

[00388] After the end of the infusion, the decline of the observed FVIII activity exhibited monoexponential decay characteristics until the baseline level was reached. The rate of decline in FVIII activity was slower for rFVIIIFc than for ADVATE* with mean (SD) model-predicted elimination half-life values of 10.7 1.98 and 10.3 3.27 hr for ADVATE* and 16.2 + 2.92 and 19.0 7.94 hr for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. Elimination half-life values appeared to be dose-independent over the dose range evaluated for both FVIII products.

[00389] Total systemic FVIII exposure (assessed by AUCINF) was ~ 53% and 84% greater following rFVIIIFc administration than ADVATE* at 25 and 65 IU/kg dose levels, respectively. Mean (±SD) model-predicted AUCNF values were 1080 i 236 and 2320 ± 784 hr*IU/dL for ADVATE and 1650 408 and 4280 1860 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

[00390] Similar to elimination half-life, the MRT was prolonged for rFVIIIFe relative to ADVATE*. Mean (SD) model-predicted MRT values were 15.3 2.86 and 14.8 +4.72 hr for ADVATE* and 23.4 4.22 and 27.3 11.4 hr for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRT values appeared to be dose-independent over the dose range evaluated for both FVIII products.

[00391] In addition, primary PK parameter values for CL and V were determined. CL values for rFVIIIFc only accounted for ~ 58-66% of those observed for ADVATE* at equivalent doses. Mean (+SD) model-predicted CL values were 2.39 0.527 and 3.21 + 1.40 mL/hr/kg for ADVATE* and 1.57 0.349 and 1.86 0.970 mL/hr/kg for rFVIIIFe for the 25 and 65 IU/kg dose groups, respectively. V values were comparable between ADVATE* and rFVIIIFe with mean (SD) model-predicted V values of 35.8 5.52 and 43.6 11.2 mL/kg for ADVATEand 35.9 6.65 and 42.7 + 8.91 mL/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. Increases in mean CL and V values were noted with increasing dose of ADVATE* and rFVIIIFe; however, the increase in standard deviations at 65 IU/kg coupled with limited dose levels confounded an assessment of the dose-dependency of these parameters.

[00392] In addition to the primary PK parameters, secondary PK parameters (e.g. K-values, IVR, etc.) were determined to evaluate FVIII duration of effect. Evidence of PK difference was also observed with rFVIIIFc demonstrating increased TBLPland TBLP3 values compared to ADVATE* at equivalent doses. IVR and K-values for ADVATE* and rFVIIIFc appeared to be comparable.

[00393] A slight increase in TBLP1 and TBLP3 values were observed with increasing dose of ADVATE* and rFVIIIFc. In contrast, slight decreases in mean IVR and K-values were noted with increasing dose of ADVATE and rFVIIIFc. As previously indicated, an assessment of the dose dependency of these parameters is confounded by limited dose levels.

[00394] Mean (SD) observed TBLPI were 2.70 ±0.511 and 3.09 ±0.978 IU/dL per IU/kg for ADVATE* and 4.06 + 0.798 and 5.66 2.38 IU/dL per lU/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. Mean (SD) observed TBLP3 were 1.98 + 0.377 and 2.39 0.718 IU/dL per IlU/kg for ADVATE* and 3.04 0.598 and 4.44 1.84 IU/dL per IU/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

[00395] Mean IVR and K-values calculated using observed C., values (subtracted with baseline and residual drug within the model) were generally greater than values determined using model-predicted C., values; consistent with slight underestimation of the observed peak activity using the one-compartment model. Mean (+SD) observed K-values were 3.08

+ 0.429 and 2.85 0.721 IU/dL per IU/kg for ADVATE*and 3.12 ±0.451 and 2.92 0.985 IU/dL per IU/kg for rFVIIIFc for the 25 and 65 U/kg dose groups, respectively. Mean (SD) observed IVR values were 112 14.5 and 116 26.9 % for ADVATE* and 113 16.3 and 117 33.6 % for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

Conclusions

[00396] All ADVATE*- and rFVIIIFc-treated patients had comparable dose-related increases in Cm. and AUCoNF over the dose range evaluated. Peak plasma levels of ADVATE* and rFVIIIFc activity were generally observed within the first hour after the end of the infusion and remained detectable for several days after dosing. After the end of infusion, the decline in baseline corrected FVIII activity exhibited monoexponential decay until the baseline was reached for both products. Parameter values for elimination half-life and MRT appeared to be dose-independent over the dose range evaluated for both FVIIl products. Slight increases in mean CL and V values were noted with increasing dose of ADVATE* and rFVIIIFc; however, increased intersubject variability at the 65 IU/kg coupled with limited dose levels confounded an assessment of the dose-dependency of these parameters.

[003971 Comparison of rFVIIIFc and ADVATESactivity PK revealed an approximate 48-61% (One-Stage Assay) or 53-84% (Chromogenic Assay) increase in systemic exposure, approximate 30-40% reduction in clearance, and an approximate 50-80% increase in both elimination half-life and MRT for rFVIIIFe relative to ADVATE" at comparable doses. Evidence of PK difference was also observed with rFVIIIFe demonstrating increased TBLP and TBLP3 values compared to ADVATE* at equivalent doses. IVR and K-values for ADVATE*and rFVIIIFc appeared to be comparable.

[00398] The PK parameters obtained from the Chromogenic Assay results generally agreed with those from the One-Stage Assay, except that the Chomogenic Assay yielded a higher estimation of exposure parameters (e.g., Cmax, AUCNF, etc.).

[00399] The observed improvements in PK, indicate that rFVIIIFc can provide prolonged protection from bleeding, allowing less frequent injections for individuals with Hemophilia A.

Example 6 rFVIIIFc A-LONG Phase 3 Clinical Study

[00400] On the basis of the interim PK analysis from the first in-human study of rFVIIIFc (see Example 3), the A-LONG study was designed. A-LONG was an open label, global, multi center, Phase 3 evaluation of the safety, pharmacokinetics, and efficacy of recombinant FVIII Fe fusion (FVIII:Fc) in the prevention and treatment of bleeding in previously treated subjects with severe hemophilia A (defined as <1 IU/dL [<%] endogenous FVIII).

[00401] The primary objectives of the A-LONG study were (i) to evaluate safety and tolerability of rFVIIIFc administered as prophylaxis, weekly, on-demand, and surgical treatment regimens, and (ii) to evaluate the efficacy of rFVIIIFc administered as tailored prophylaxis, on-demand, and surgical treatment regimens. The secondary objective of the A LONG study were (i) to characterize the PK profile of rFVIIIFc and compare the PK of rFVIIIFc with the currently marketed product, ADVATE*, (ii) to evaluate individual responses with rFVIIIFc, (iii) to characterize the range of dose and schedules required to adequately prevent bleeding in a prophylaxis regimen, maintain homeostasis in a surgical setting, or to treat bleeding episodes in an on-demand, weekly treatment, or prophylaxis setting, and (iv) to evaluate rFVIIIFc consumption (e.g., total annualized rFVIIIFc consumption per subject).

[004021 165 subjects were enrolled into one of three regimens: a tailored prophylaxis regimen (Arm 1), a weekly dosing regimen (Arm 2), and an on-demand regimen (Arm 3). In addition, rFVIIIFc was evaluated in a perioperative management subgroup.

[00403] Key inclusion criteria: (i) male, (ii) 12 years of age and at least 40 kg, (iii) diagnosis of severe hemophilia A defined as <1% (<1 IU/dL) endogenous FVIII activity, and (iv) history of >150 prior documented exposure days with any currently marketed FVIII product.

Arm 1: Tailored Prophylaxis Regimen 100404] Arm1 included an overall group and a PK subgroup. The initial regimen was twice weekly at 25 IU/kg on the first day, followed by 50 IU/kg on the fourth day of the week (Day 4). Subjects were administered rFVIIIFc on this weekly prophylaxis regimen until PK results for rFVIIIFc were available. Based on these results, a tailored prophylaxis regimen was established for each individual, in which the dose and interval was determined to maintain a trough level of 1-3% FVIII activity. Each subject was then administered his individually tailored prophylaxis regimen throughout the study. 100405] Subjects were monitored throughout the study and ongoing dose and interval adjustments were made. Adjustments were only made when a subject experienced unacceptable bleeding episodes defined as 2 spontaneous bleeding episodes over a rolling two-month period. In this case, adjustment targeted trough levels of 3-5%.

Arm 2: Weekly Dosing Regimen

[004061 Subjects underwent abbreviated rFVIIIFc PK profiling as follows: Washout of at least 96 hours; a single dose of rFVIIIFc 65 IU/kg; Abbreviated sampling beginning on rFVIIIFc Day 0, including pre-injection and 10 (±2) minutes, 3 hours ( 15 minutes), 72 ( 2) hours

[Day 3], and 96 (± 2) hours [Day 4] from the start of injection. Following the abbreviated PK profiling, subjects were then administer a fixed dose of 65 IU/kg rFVIIIFc every 7 days at least for 28 weeks and up to 52 weeks.

Arm 3: Episodic (On-demand) Treatment

[00407] Subjects received rFVIIIFc episodic treatment as needed for bleeding. Subjects were enrolled and randomized and underwent abbreviated rFVIIIFc PK profiling as follows: (i) Washout: At least 96 hours.

(ii) Dosing at rFVIIIFc Day 0: A single dose of rFVIIIFc 50 IU/kg administered under medical supervision. (iii) Abbreviated sampling beginning at rFVIIIFc Day 0: Preinjection and 30 (+3) minutes, 3 hours (±15 minutes),. 72 (2) hours [Day 3], and 96 (2) hours [Day 4] from the start of the injection.

[00408] At the selection sites, sampling for TGA was done coincident with all PL profiling time points. For the subset of subjects undergoing sampling for ROTEM/TEG, collections were done at the following time points: Preinjection and 3 hours (± 15 minutes), 72 (±2)hours

[Day 3], and 96 (+2) hours [Day 4] from the start of the injection.

1004091 Between scheduled visits, the subjects treated bleeding episodes at rFVIIIFc doses between 10 and 50 IU/kg, depending on the severity of the bleeding.

Perioperative Management Subgroup

[004101 rFVIIIFc was administered prior to a following surgery in the subset of patients requiring a major surgical procedure during the study. Major surgery is defined as any surgical procedure (elective or emergent) that involves general anesthesia and/or respiratory assistance in which a major body cavity is penetrated and exposed, or for which a substantial impairment of physical or physiological functions is produced (e.g., laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation).

1004111 For prophylaxis during surgery, subjects were treated with 20 to 50 IU/kg rFVIIIFc every 12 to 24 hours. Prior to surgery, the physician reviewed the subject's rFVIIIFc PK profile and assessed the dose regimen of FVIII replacement generally required for the type of planned surgery and the clinical status of the subject. Recommendation for the appropriate dosing of rFVIIIFc in the surgical treatment period, including any rehabilitation time, took these factors into consideration.

[00412] Pharmacokinetic (PK) Assessment: All subjects in all arms had an initial PK assessment after their first dose of rFVIIIFc. A subset of subjects from Arm 1 were assigned to a protocol-specified sequential PK subgroup to compare the PK of rFVIIIFc with recombinant factor VIII (rFVIII, ADVATE® [anti-hemophilic factor (recombinant) plasma/albumin-free method]) as follows: (i) Prior to treatment in Arm 1, PK was assessed after a single dose of ADVATE® 50 IU/kg. PK was then assessed in these same subjects after a single dose of rFVIIIFc 50 IU/kg.

(ii) PK of rFVIIIFc was repeated at 12 to 24 weeks.

100413] Key efficacy outcome measures (included in initial readout): (i) Annualized bleeding rate (ABR) in Arm 1 versus Arm 3 (individualized prophylaxis arm compared with episodic treatment arm), (ii) number of injections required to resolve a bleeding episode, (iii) treating physicians' assessments of subjects' response to surgery with rFVIIIFc using a 4 point scale. 1004141 PK outcome measures: PK of rFVIIIFc and ADVATE*.

[00415] Key safety outcome measures: (i) Incidence of inhibitor development; and, (ii) incidence of adverse events (AEs) occurring outside of the perioperative management period.

Results:

[00416] Subjects: A total of 165 subjects were enrolled in the study. Arm 1 (individualized prophylaxis), n = 118; Arm 2 (weekly prophylaxis), n = 24; Arm 3 (episodic treatment), n = 23; Perioperative management subgroup, n = 9, 9 surgeries (8 subjects from Arn 1, and 1 from Arm 2). 92.7% of subjects completed the study.

[00417] Efficacy: Median ABR (individualized prophylaxis arm: 1.6; weekly prophylaxis arm: 3.6; episodic treatment arm: 33.6). In the individualized prophylaxis arm, the median dosing interval was 3.5 days during the last 3 months on study. 30 percent of patients in the individualized prophylaxis arm achieved a mean dosing interval of at least 5 days. 98% of bleeding episodes were controlled by one or two injections of rFVIIIFc. In perioperative management, treating physicians rated the hemostatic efficacy of rFVIIIFc as excellent or good in 100% of surgeries.

[00418] PK: The geometric mean terminal half-life of rFVIIIFc was approximately 19.0 hours, which is 1.53-fold longer than that of ADVATE* (approximately 12.4 hours).

[00419] Safety: No inhibitors were detected to rFVIIIFc, and no cases of anaphylaxis were reported. rFVIIIFc was generally well tolerated. The most common AEs, regardless of causality, (incidence >5%) occurring outside of the perioperative management period were nasopharyngitis, arthralgia, headache, and upper respiratory tract infection. 12 subjects (7.3%) experienced at least one serious AE (SAE) outside of the perioperative management period. No SAEs were assessed to be related to drug by the investigator.

Summary

[00420] Individualized and weekly prophylactic regimens resulted in low single-digit median annualized bleeding rates. In the individualized prophylaxis arm, the median dosing interval was 3.5 days. During the last 3 months on study, 30 percent of patients in the individualized prophylaxis arm achieved a mean dosing interval of at least 5 days. 98% of bleeding episodes were controlled by one or two injections of rFVIIIFc. Hemostatic efficacy of rFVIIIFc during surgery was rated by treating physicians as excellent or good in 100% of surgeries. The half life of rFVIIIFc was approximately 19.0 hours compared to 12.4 hours for ADVATE*. No subject developed an inhibitor or experienced an anaphylactic reaction to rFVIIIFc. Recombinant FVIIIFc was generally well tolerated.

Example 7 Clinical ROTEM* Assessment

[00421] In addition to the measurement of plasma FVIII activity by one-stage activated partial thromboplastin time (aPTT) assay, whole blood rotational thromboelastometry (ROTEM*) has also been explored to assess the improvement in global hemostasis by rFVIIIFc and ADVATE* in 2 subjects, specifically, 1in the low dose cohort and 1 in the high dose cohort.

[004221 rFVIIIFc and ADVATE* appear to be comparably active in clot formation when spiked into subjects' blood prior to rFVIIIFc treatment. The clotting time (CT) was linear with respect to the dose of rFVIIIFc and ADVATE* in the range of approximately 1% of 100% of normal, and the dose response was comparable between rFVIIIFc and ADVATEin the same subject.

[00423J Following dosing with ADVATE*and subsequently rFVIIIFc, citrated whole blood was sampled at various time points and the clot formation following recalcification was monitored by ROTEM*. Despite the variable baseline CT due to residue FVIII levels prior to ADVATE* or rFVIIIFc dosing, both products effectively corrected the CT to comparable levels 30 minutes post-injection. In addition, the improvement in CT was better sustained at and after 3 hours post-injection of 25 IU/kg of rFVIIIFc relative to ADVATEOin the subject dosed at this low dose. However, the differential improvement of rFVIIIFc versus ADVATE* was much less appreciable at the 65lU/kg dose.

Example 8 In vivo Efficacy of rFVHIFc and Single Chain (SC) rFVI in HemA Mice

[004241 Recombinant Factor VIIFc (rFVIIIFc) is comprised of a B domain deleted (BDD) rFVIII protein genetically fused to the Fe domain of human immunoglobulin GI (IgG). Prior to secretion from HEK 293 cells, most of the rFVIIIFc is processed into a FVIII heavy chain (HC) and light chain (LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrand factor (VWF) and released upon activation in a manner that is indistinguishable from native FVIII. Spontaneous dissociation of the HC and LC is thought to contribute to the loss of FVIII activity in plasma and during storage of FVIII drug products. Here we describe a single chainnon-processed isoform of rFVIIIFc (SC rFVIIIFc), which can provide superior manufacturability and enhanced stability compared to native FVII. 1004251 SC rFVIIIFc was purified from rFVIIIFe, which contains a fraction of the non processed isoform. Compared to rFVIIIFe, SC rFVIIIFc showed equivalent chromogenic activity but approximately 60% reduced activity by the one stage (aPTT) assay, (TABLES 3A and 3B). Thrombin generation assay (TGA) was performed using calibrated automated thrombogram (from Thrombinoscope*). In a thrombin generation assay (TGA), SC rFVIIIFc also showed a reduced thrombin potential (FIG. 13A), and peak thrombin (FIG. 13B) compared to rFVIIIF. However, as shown in TABLE 3B, full activity of SC rFVIIIFc by aPTT was observed in the absence of vWF, suggesting release from vWF can be delayed due to covalent linkage of the a3 acidic region to the HC after Arg 1680 cleavage in SC rFVIIIFc, in contrast to a3 release and dissociation from fully processed FVII. Delayed dissociation from vWF can explain the reduced activity observed in the aPTT assay and TGA, while full activity was observed in the two-stage chromogenic assay. A reduced rate of activation in the presence of vWF was confirmed in a modified chromogenic substrate assay with limiting thrombin as FVIII activator.

[00426] In vivo function of SC rFVIIIFc was assessed in the HemA mouse tail vein transection (TVT) model. The mice were first anesthetized and then injected with 4.6 g/kg, 1.38 g/kg, or 0.46 g/kg of either processed rFVIIIFe (Drug Substance, which contain about 75%-85% processed rFVIIIF) and purified single chain (SC) rFVIIIFc 48 hours prior to TVT. The tail was cut from the tip and immediately placed into a tube to collect blood. Percentage of protection on survival was measured for rFVIIIFc processed (drug substance) and single chain (SC) FVIIIFc as shown in TABLE 7 and FIGS. 7A, 7B and 7C.

TABLE 7. In vivo Efficacy of rFVIIIFc DS and Single Chain (SC) rFVIIIFc

Dose (pg/kg) 4.6 1.38 0.46 % of Protection FVIIIFc DS 93 52 19 on Survival Single chain 93 64 14 rFVIIIFc

1004271 In vivo Tail re-bleeding and survival were monitored hourly up to 12 hours post TVT with final observation performed at 24-hour post TVT. SC rFVIIIFc and the rFVIIIFc demonstrated equivalent in vivo efficacy in this model, with an ED50 of 1.17 pg/kg and 1.23 ig /kg respectively when TVT was performed at 48 hours post infusion (FIG. 7A). Comparable 24 hour post TVT survival curves (p > 0.65) (FIG. 713) and re-bleed rates (FIG. 7C) in HemA mice were observed for the SC rFVIIIFc and rFVIIIFc at each tested dose level, indicating that SC rFVIIIFc was equally effective as rFVIIIFc despite its lower apparent aPTT activity. The delayed in vitro activation of SC rFVIIIFc in the presence of vWF therefore appeared to have no significant impact on its in vivo efficacy. These observations indicated that SC rFVIIIFc represents a novel and efficacious isoform of rFVIIIFc with potential clinical applications.

Example 9 Phase 1/2a study Clinical Trial

[00428] rFVIIIFe is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fe domain of human IgG1 to extend circulating rFVIII half-life. This first-in-human study in previously-treated male subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of ADVATO at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-FVIIIFc antibodies or inhibitors. Across dose levels, as compared with ADVATE* rFVIIIFc showed 1.54 to 1.71-fold longer elimination t/2 and mean residence time, 1.49 to 1.56-fold lower clearance, and 1.48 to 1.56-fold higher total systemic exposure. ADVATE* and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was approximately 1.53 to 1.68-fold longer than ADVATE* across dose levels. Thus, rFVIIIFc can offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A.

[00429] rFVIIIFc is a recombinant fusion protein composed of a single molecule of B-domain deleted rFVIII covalently linked to the human IgG1 Fe domain. Potential advantages of Fc fusion proteins include better tolerability and prolonged hemostatic protection, and the Fe domain represents a natural molecule with no known inherent toxicity. Dumont J.A. et al,BioDrugs20(3):151-60 (2006), Dumont J.A. et al, "Monomeric Fe fusion technology: an approach to create long-lasting clotting factors," in: Kontermann R., ed., Therapeutic Proteins- Strategies to Modulate Half-Life, Chapter 11, Wiley VCH publisher; prepublished online, DOI: 10.1002/9783527644827.chlO. Attachment to the IgG, Fe domain permits binding to the neonatal Fc receptor (FcRn), which is expressed in many cell types, including endothelial cells. FeRn expression remains stable throughout life and is responsible for protecting IgG 1 and Fc-fusion proteins from lysosomal degradation, thus prolonging the ti2 of the protein. Dumont J.A. et al., BioDrugs20(3): 151-60 (2006), Roopenian D.C. et al., Nat Rev Immunol. 7(9):715-25 (Epub 2007 Aug 17). Numerous proteins within the circulation are internalized into the cells lining the vasculature via nonspecific pinocytosis and are trafficked to endosomal and lysosomal degradation pathways. 100430] Fc proteins interact with FeRn, resident within endosomes. Endosomes containing FcRn direct the Fe fusion proteins back to the plasma membrane, releasing them into circulation in a pH-dependent manner, Lencer W.I. and Blumberg R.S., Trends Cell Biol. 15(1):5-9 (2005) thereby avoiding lysosomal degradation. This recycling approach has been used successfully to extend the t/ of therapeutic biologics; a number of Fc fusion-based drugs have been approved for clinical use (e.g., etanercept, romiplostim) and others are in development. Huang C., Curr Opin Biotechnol. 20(6):692-9. (Epub 2009 Nov 4), Schmidt S.R., CurrOpin DrugDiscov Devel. 12(2):284-295 (2009).

[00431] Preclinical data for rFVIIIFc indicate that FVIII can be rescued from degradation by a natural protective pathway mediated by FcRn, thus extending t1/. In Hemophilia A mice and dogs, terminal plasma t1 for rFVIIIFe was approximately 2 times longer than with rFVIII. Dumont J.et aL, Blood. 116(21) Abstract 545 (2009), Liu T. et al., J Thromb Haemost. 9(S2):561 (2011). Based on these data, we conducted a first-in-human clinical study to investigate the safety and PK of a long-lasting rFVIIIFc fusion protein in subjects with hemophilia A. Study Design

[00432] This open-label, dose-escalation, multicenter Phase 1/2a study in previously treated patients with severe hemophilia A investigated the safety of rFVIIIFc and its pharmacokinetics (PK) compared with ADVATE* (antihemophilic factor [recombinant], plasma/albumin-free method, octocog alfa, Baxter Healthcare). This study was performed in accordance with the US CFR and ICH Guidelines on Good Clinical Practices. Prior to any testing, approval from participating Institutional Review Boards and written informed consents from all subjects were obtained. The study design was sequential; a single dose of ADVATE* was administered at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc (Figure 8). Both drugs were injected intravenously over approximately 10 minutes. The two dose levels were expected to bracket the typical therapeutic dose ranges. Subjects were followed for 28 days after receiving rFVIIIFc for safety analyses, including testing for anti-FVIII antibodies and inhibitors at 14 and 28 days post-injection. Plasma FVIII activity was measured in subjects before injection, 10 and 30 minutes, 1, 3, 6, 9, 24, 48, 72, 96, 120, and 168 hours (7 days) after rFVIIIFc injection, with additional samples at 192, 216, and 240 hours (10 days) for subjects dosed at 65 IU/kg of rFVIIIFc. Plasma FVIII activity was measured at the same time points after ADVATE* treatment, through 72 hours for the 25 IU/kg group and 96 hours for the 65 IU/kg group. (a) Subjects

[00433] Male subjects were at least 12 years of age with severe hemophilia A (defined as FVIII activity level < 1%) and had at least 100 documented prior exposure days to FVIII concentrates (pdFVIII or rFVIII). Subjects with known hypersensitivity to mouse or hamster protein, history of inhibitor or detectable inhibitor titer at screening, or who were taking any medications that could affect hemostasis or systemic immunosuppressive drugs, or who experienced an active bacterial or viral infection (other than hepatitis or HIV) within 30 days of screening were excluded. Subject's genotype was recorded at study entry, when known. (b) Treatment Product

[004341 The human rFVIIIFc and Fe transgenes were stably transfected into HEK293 cells and the cell line was extensively tested for stability, sterility, and viral contamination to ensure safety. The purified drug product is composed of a monomeric B-domain-deleted FVIII covalently linked through its carboxy-terminus to the N-terminus of an Fe monomer, which forms a disulfide bond with a second Fe monomer during synthesis and secretion from the cells. rFVIIIFc was purified by chromatography and nanofiltration, and was fully active in one-stage and chromogenic clotting assays relative to commercially available rFVIII preparations. It was supplied as a frozen liquid containing 1000 IU per 2 mL of solution and formulated with L-histidine (pH 7), sodium chloride, calcium chloride, sucrose, mannitol, and Polysorbate 20. For injection, the product was diluted with saline solution (0.9% NaCl). (c) Outcome Measures

[004351 The primary objective of the study was safety, evaluated through physical examination, reporting of treatment-emergent adverse events (AEs), development of antibodies, and laboratory monitoring over time. The secondary objectives included parameters derived from PK analyses. Laboratory assessments included prothrombin time, activated partial thromboplastin time (aPTT), international normalized ratio, levels of D-dimer, von Willebrand factor (vWF) antigen, standard hematology and blood chemistry tests, and urinalysis.

[00436] FVIII activity was measured by the one-stage clotting (aPTT) assay on a Siemens BCS-XP analyzer using commercial reagents (Dade Actin FSL) with calibration against a normal reference plasma (Precision Biologics CRYOcheck") traceable to the World Health Organization (WHO) 5th International Standard (IS) for human plasma. In addition to the aPTT assay, FVIII activity was measured by a chromogenic substrate assay Rosen S., ScandJ Haematol Suppl. 33(Suppl 40):139-45 (1984) using a commercially available kit (Aniara BIOPHEN FVIII:C) that complies with European Pharmacopoeia recommendations. The chromogenic assay was calibrated against normal human reference plasma (Instrumentation Laboratories ORKE45), which also had a potency assigned against the WHO 5th IS human plasma standard.

[004371 The lower limit of quantification (LLOQ) for the one-stage and chromogenic assays was 0.5 IU/dL and 0.4 IU/dL, respectively. FVIII inhibitors were measured by the Nijmegen-modified Bethesda assay and less than 0.6 BU/mL was considered negative. Anti rFVIIIFc antibodies were assessed using a specific bridging electrochemiluminescent immunoassay which uses biotin and sulfo-tagged rFVIIIFc. Assay sensitivity was determined to be 89 ng/mL using an anti-human FVIII monoclonal antibody as a surrogate control. Exploratory whole blood rotation thromboelastometry (ROTEM*) was performed in two subjects, one from each dose level, at various time points to assess the improvement in global hemostasis following injection with ADVATE* and rFVIIIFc. (d) Pharmacokinetic Analyses

[004381 A user-defined one-compartment disposition model, which automatically estimates the endogenous FVIII level and subsequent residual decay, was utilized in WINNONLIN* for analysis of the individual subject plasma FVIII activity-versus-time data following a single administration of ADVATE* or rFVIIIFec. Actual sampling times, doses, and duration of injection were used for calculations of parameters including maximum activity (Cma), t 2 , clearance (CL), volume of distribution at steady-state (V,,), area under the curve (time zero extrapolated to infinity [AUCINF), mean residence time (MRT), and incremental recovery.

[00439 Monte Carlo Simulation of rFVIIFc Activity-Versus-Time Profile: To construct FVIII activity-time profiles following dosing regimens of 25 IU/kg or 65 IU/kg, a Monte Carlo simulation was conducted using the population PK model of ADVATE* and rFVIIIFc. The mean estimates of model parameters (CL, volume of distribution) in the tested population, the inter-individual variance, and the residual variability were estimated based on the one-stage (aPTT) clotting assay activity data of ADVATE* and rFVIIIFc from 16 subjects in this Phasel/2a study. Five hundred subjects were simulated with 15 sampling points for each subject for each dosing regimen. The percentage of the population with FVIII activity above or equal to 1% and 3% at different times following different dosing regimens of ADVATE* or rFVIIIFc was estimated.

[004401 StatisticalAnalyses: Selected PK parameters for rFVIIIFc and ADVATE* were compared using an analysis of variance model. PK parameters were log-transformed for these analyses and estimated means, mean differences, and confidence intervals on the log-scale were transformed to obtain estimates for geometric means, geometric mean ratios (GMR), and confidence intervals, respectively, on the original scale. The GMR is the geometric mean of the intra-subject ratio of the rFVIIIFc PK parameter value to the ADVATE PK parameter value.

Results

[004411 Subject Disposition: Nineteen subjects were enrolled in the study; 16 underwent PK evaluation for both ADVATE* and rFVIIIFc. One subject self-administered his previous product prior to completing the wash-out period following the dose with ADVATE* and was thus excluded from the PK analysis, but was included in the safety analysis. Three subjects were discontinued from the study before receiving either study drug: one voluntarily withdrew; a second was withdrawn by the Investigator for non-compliance; and one was withdrawn at the Sponsor's request due to completion of study enrollment. Of the subjects dosed, six subjects received 25 IU/kg and 10 subjects received 65 IU/kg of both ADVATE* and rFVIIIF. Mean age was 40.3 years (23 to 61 years). Genotypic identification was collected for seven subjects; inversion of intron 22 was reported in six subjects; and a frame-shift defect was reported in one subject. The genotype was unknown for nine subjects. Thirteen subjects had hepatitis C antibodies, four of whom were also positive for HIV.

[00442] Safety: Forty-four treatment-emergent AEs were reported by 11 (69%) subjects during the treatment and follow-up periods. This included the day of dosing with ADVATE* or rFVIIIFc through a 28-day post-dosing observation period. The majority of events were considered mild and none led to withdrawal from the study. One event, dysgeusia, occurred transiently in one subject while receiving a 65 IU/kg dose of rFVIIFc and was considered related to rFVIIIFc. One subject experienced an anxiety attack after receiving 65 IU/kg of rFVIIIFc which resulted in 21 AEs, 19 of which were graded as mild, and two of which (headache and photophobia) were rated as moderate. Neither was deemed related to rFVIIIFc by the Investigator. No serious bleeding episodes were reported. No evidence of allergic reactions to injection was detected. All plasma samples tested negative for FVIII inhibitors and anti-rFVIIIFc antibodies. No signs of injection site reactions were observed. No clinically meaningful changes in abnormal laboratory values were reported.

[004431 Pharmacokinetics:

[00444] Correlation Between aPTT and Chromogenic Activity for rFVIIIFc in Plasma: ADVATE* and rFVIIIFc activities were determined in the same assays using commercially available reagents and calibration against normal human plasma standards. There was a strong correlation between the results obtained by the one-stage clotting assay and the chromogenic assay in samples that had an activity above the LLOQ. Correlation coefficients (Pearson R2) of 0.94 and 0.95 were observed between the two assay results for 151 samples following ADVATE* dosing and 185 samples following rFVIIIFc dosing, respectively. Compared to the aPTT results, the chromogenic FVIII activities were, on average, 21% higher for ADVATE' and 32% higher for rFVIIIFc, not statistically significant (FIG. 9). This observation led to a slightly higher estimation of exposure parameters by the chromogenic assessment for both drugs. The apparent higher FVIII recoveries by the chromogenic assay are typical for recombinant FVIII products tested in clinical assays, and are in agreement with most other marketed FVIII products. Lee C.A. et al., Thromb Haemost. 82(6):1644-7 (Dec. 1999), Mikaelsson M. and Oswaldsson U., Semin Thromb Hemost. 28(3):257-64 (June 2002), Stroobants A.K. et al, J Thromb Haemost. 9 (Suppl 2) (2011). 100445] Improved Pharmacokineticsfor rFVIIIFc: The primary PK estimates were derived from one-stage (aPTT) clotting assay activity data. In subjects who received 25 or 65 IU/kg of ADVATE* followed by an equal dose of rFVIIIFc, the plasma FVIII activity rose sharply and reached Ca within the first hour following dosing. The subsequent decline of the observed FVIII activity exhibited monoexponential decay characteristics until the baseline

FVIII activity was reached (FIGs. 10A and 10R). The C. increased proportionally to the dose, but was comparable between equal doses of ADVATE* and rFVIIIFc (TABLE 8). The total exposure (AUCINF) also increased proportionally to the dose. However, the AUCINF of rFVIIIFe was 1.48 and 1.56-fold greater than that of ADVATE* at 25 IU/kg (p0.002 ) and 65 IU/kg (p<0.001), respectively (TABLE 8).

TABLE 8. PK Parameters by One-Stage (aPTT) Assay for rFVIIIFc and ADVATE* Per Dose Group

Paramete Dose: 25 IU/kg (N=6) Dose: 65 IU/kg (N=9) r ADVATE rFVIIIFc Geom. ADVATE*' rFVIIIFc Geom. * Geom. Mean Ratio Geom. Geom. Mean Mean Ratio Geom. Mean [95% CI] Mean [95% C] [95% CI] Mean [95% CI] (p-value) [95% CI] (p-value)

[95% CI] C._OB 0.952 0.895 S 63.6 60.5 [0.819, [0.795, (IU/dL) [59.1, [53.1, 1.11] 133 119 1.01] 1 68.3] 69.0] (p = 0.440) [105,168] [103, 136] (p = 0.061) AUCINF 994 1480 1.48 1800 1.56 (hr*IU/d [723, [1160, [1.26,1.76] [1350, 2803970] [1.33, 1.83] L) 1370] 1880] (p = 0.002) 2400] (p < 0.001) 12.2 18.8 1.54 11.0 18.8 1.70 tm (hr) [9.14, [14.8, [1.40,1.69] [8.76 13.9] [14.3,24.5] [1.54,1.89] 16.3] 23.8] (p <0.001) (p < 0.001) 17.5 27.0 1.54 15.8 27.01.71 MRT (hr) [13.1, [21.3, [1.40,1.69] [12.6,19.9] [20.6 35.3] [1.54,1.89] 23.4] 34.2] (p< 0.001)<0.001) CL 2.49 1.68 0.673 0.642 (mL/hour [1.80, [1.31, [0.569, 3.61 2.32 [0.547, /kg) 3.45] 2.15] 0.796] [2.71, 4.83] [1.64,3.29] 0.753] (p = 0.002) (p <0.001) V 43.9 45.4 1.04 57.4 62.8 1.09 (VL/kg) [39.3, [39.3, [0.947,1.13] [48.3, 68.3] [55.2, 71.5] [0.976,1.22] 49.0] 52.5] (p = 0.357) (p = 0.107) Incremen tal 2.56 2.44 0.952 0.894 Recovery [2.36, [2.12, [0.819,1.11] 2.04 1.183 [0.795,1.01] (IU/dL 2.78] 2.81] (p = 0.444) [1.61, 2.59] [1.59, 2.10] (p= 0.060) per IU/kg)

CI= Confidence Interval; Geom. Mean = Geometric Mean; OBS = observed. Estimated means, 95% CI for means, and mean differences were transformed to obtain estimated geometric means, 95% CI for geometric means, and geometric mean ratios, respectively.

1004461 The t12, MRT, CL, and V,, appeared to be independent of dose (TABLE 8). The geometric mean t12 of rFVIIIFc was 18.8 hours for both the 25 IU/kg and 65 IU/kg dose groups. This represents a 1.54 and 1.70-fold improvement over that of ADVATE* (12.2 hours and 11.0 hours) at equivalent doses (p<0.001), respectively (TABLE 8). The same intra-subject improvement was observed in the MRT of rFVIIIFe (27.0 hours for both dose groups) compared with ADVATE* (17.5 hours for the 25 IU/kg and 15.8 hours for the 65 IU/kg) (p<0.001). Consistent with improvement in the t2 and MRT was a corresponding 1.49 and 1.56-fold reduction in intra-subject CL at doses of 25 IU/kg (p=0.002) and 65 IU/kg (p<0.001), respectively. There were no significant differences in V,, and incremental recovery between ADVATE* and rFVIIIFc. Therefore, within each subject, rFVIIIFc demonstrated an improved PK profile compared with ADVATE*. 1004471 The improved PK profile of rFVIIIFc resulted in increased time post-dosing to 1% FVIII activity which was 1.53 and 1.68-fold longer respectively, than with ADVATE* at 25 IU/kg (p<0.001) and 65 IU/kg (p<0.001) (data not shown), suggesting a potentially longer therapeutic duration for rFVIIIFe. The favorable PK profile of rFVIIIFc relative to ADVATE was also demonstrated by FVIII activity measured in the chromogenic assay (TABLE 9), which was comparable to data derived from aPTT assays. The estimation of exposure, i.e., Cm. and AUCNF, was slightly higher, however, based on the chromogenic assay than on the one-stage (aPTT) clotting assay for both ADVATEO and rFVIIIFc.

TABLE9 PK Parameters by Two-Stage (Chromogenic) Assay for rFVIIIFc and ADVATE* Per Dose Group Parameter Dose: 25 IU/kg (N=6) Dose: 65 lU/kg (N=9) ADVATE* rFVIIIFc Geom. ADVATE rFVIIIFc Geom. Geom. Geom. Mean Mean Ratio * Geom. Mean Ratio Mean [95% CI] [95% CI] (p- Geom. Mean [95% CI]

[95% CI] value) Mean [95% CI] (p-value)

[95% CI] CmiaxOB 75.5 1.01 1.04 S [65.5, 76.5 [0.940,1.09] 175 182 [0.900, (IU/dL) 87.1] [64.9, 90.1] (p = 0.686) [143, 215] [146, 227] 1.20}

(p = 0.571) AUCINF 1060 1.57 2270 4280 1.89 (hr*IU/d [822, [1300,2120] [1.38, 1.80] [1670, [2960, [1.61, 2.21] L) 1360] [U(p < 0.001) 3070] 6190] (p < 0.001) 10.5 16.7 1.59 10.8 19.8 1.84 tU (hr) [8.49, [13.8, 20.1] [1.35, 1.87] [8.16, [14.3, [1.60,2.12] 12.9] (p<0.001) 14.2] 27.5] (p < 0.001) 15.0 23.9 1.59 15.4 28.5 1.85 MRT (hr) [12.2' [19.8, 28.9] [1.35, 1.87] [11.7, [20.5, [1.61, 2.12] 18.6] (p < 0.0 0 1) 20.4] 39.6] (p < 0.001) CL 2.35 0.636 2.87 1.52 0.530 (mL/hour [1.80, 1.49 [0.557, [2.12, [1.05, [0.453, /kg) 3.06] [1.16, 1.92] 0.727] 3.89] 2.20] 0.620] (p< 0.001) (p < 0.001) 35.5 35.9 1.01 44.5 43.4 0.975 Vss (mL/kg) [30.5, [30.4, 42.3] [0.898, 1.14 [36.7, [38.2, [0.863,1.10] 41.3] (p = 0.822) 54.1] 49.4] (p = 0. 6 5 3

) Increment al 3.05 1.01 2.70 2.80 1.04 Recovery [2.62, 3.09 [0940, 1.091 [2.20, [2.24, [0.900,1.20] per 3.54] (p = 0.679) 3.31] 3.50] (p = 0.571) IU/kg) CI= Confidence Interval; Geom. Mean =Geometric Mean; OBS = observed. Estimated means, 95% CI for means, and mean differences were transformed to obtain estimated geometric means, 95% CI for geometric means, and geometric mean ratios, respectively.

[004481 Correlation Between von Willebrand Factor and Disposition of rFVIIIFc: Because the majority of FVIII in circulation is in complex with VWF, Lenting P.J. et al., J Thromb Haemost. 5: 1353-60 (2007) and because the genome-wide association study has identified that the genetic determinants of FVIII levels are primarily dependent on VWF levels, Smith N.L. et al., Circulation 121:1382-1392 (2010) we examined the association between VWF and rFVIIIF. A strong correlation was observed between VWF levels and CL and t1 for both rFVIIIFc and ADVATF* As shown in FIGs. 11A and 11B, as the level of VWF increased, the CL of rFVIIIFc (p=0.0016) and of ADVATE* (p=0.0012) decreased.

[00449] The opposite relationship was observed between the level of VWF and t1 2 . As the level of VWF increased, the t of rFVIIIFc (p=0.0003) and of ADVATE" (p<O.OO0) increased. This correlation indicates that the Fe moiety of rFVIIIFc does not alter the role of VWF in protecting FVIII from clearance.

[00450] Effects of Prolonged PK of rFVIIIFc on Whole Blood ROTEM*: Prior to administration of study drug, blood from one subject in each dose group was spiked with an equal dose of rFVIIIFc or ADVATE* and analyzed by whole blood ROTEM*. Clotting time (CT) was linear with respect to the dose in the range of approximately 1% to 100% of normal, and the dose response was comparable between rFVIIIFc and ADVATE* in the same subject (data not shown), indicating comparable potency of rFVIIIFc and ADVATE* in clot formation. 100451] Despite the variable baseline CT due to residual FVIII levels prior to the administration of ADVATE* or rFVIIIFc, both products effectively corrected the CT to comparable levels 30 minutes post-dosing (see FIG. 12A and 12B). The improvement in CT was better sustained by rFVIIIFc than ADVATE after 3 hours following a dose of 25 IU/kg (FIG. 12A), and after 24 hours following a dose of 65 IU/kg (FIG. 12B).

[004521 rFVIIIFc was well tolerated by subjects at both doses. There were no clinically significant changes observed in hematology, blood chemistry, or urinalysis parameters. The majority of AEs were mild, unrelated to rFVIIIFe, and resolved without sequelae. No serious AEs or deaths occurred during the study, and no subjects at either dose developed neutralizing or binding antibodies to rFVIIIFe.

[00453] rFVIIIFc demonstrated a significantly improved FVIII activity PK profile relative to ADVATE*, with tv2 and MRT across dose levels being 1.54 to 1.71-fold longer, as measured by the one-stage (aPTT) clotting assay and 1.59 to 1.84-fold longer by the two-stage chromogenic assay. The prolonged activity of rFVIIIFc predicts possible prolonged efficacy, allowing for a less frequent dosing regimen in the prophylactic treatment of patients with Hemophilia A.

[00454] Adopting the PK parameters derived from this study, the Monte Carlo simulation predicted that a higher percentage of patients receiving rFVIIIFc will sustain FVIII levels above 1% or 3% as compared with patients receiving equal doses of ADVATE* (TABLE 10). For example, at a dose of 25 IU/kg, 12.2% of ADVATE* patients versus 71.2% of rFVIIIFe patients were predicted to have FVIII trough levels above 1% on Day 4; at a dose of 65 IU/kg, 11.0% of ADVATE* patients versus 66.4% of rFVIIIFc patients are predicted to have FVIII levels above 3% on Day 4. Clinical trials in larger numbers of patients are planned to confirm results from this Phase 1/2a study and from the Monte Carlo simulation predictions.

TABLE 10. Predicted Percentage of Subjects Achieving FVIII Trough Levels Above 1% and 3% of Normal at a Specified Dose Regimen of ADVATE* or rFVIIIFc

Timepoint ADVATE' rFVIIIFc following dosing (Day) 25 IU/kg 65 IU/kg 25 IU/kg 65 IU/kg Percent of Subjects with FVIII Trough Levels above 1%

3 40.0 67.8 92.6 99.0 4 12.2 31.0 71.2 90.0 5 4.20 13.6 39.4 71.6 7 0.200 1.40 7.80 26.4 Percent of Subjects with FVIII Trough Levels above 3% 3 10.6 34.6 62.2 91.0 4 1.60 11.0 25.4 66.4 5 0.200 3.20 7.00 36.2 7 0 0.200 0.400 6.60

[004551 In vitro coagulation assays demonstrate no loss of specific activity for rFVIIIFc, compared to B-domain deleted or native FVIII, by either clotting or cbromogenic assays, using commercially available reagents and commonly used FVIII reference standards (Dumont et al, Blood (2012), prepublished online DOI:10.1182/blood-2011-08-367813). In addition, these results indicate that rFVIIIFe can be reliably assayed in a clinic setting by either the one-stage assay or the chromogenic method.

[00456] In summary, this Phase 1/2a clinical trial has demonstrated the safety and prolonged t 1 of rFVIIIFc in patients with severe hemophilia A. A pivotal Phase 3 study is ongoing with

rFVIIIFc to establish effective prophylaxis dosing regimens for individuals with hemophilia A.

Example 10 Pharmacokinetics and Efficacy of rFVIIIFc in Mouse and Dog Models of Hemophilia A 1004571 The pharmacokinetics and efficacy of rFVIIIFc compared to rFVIII was evaluated in mouse and dog models of hemophilia A, in support of human studies. rFVIIIFc is a heterodimeric protein comprising a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human immunoglobulin G1 (IgG1). Traditional dimeric

Fc fusions, created through the fusion of the monomeric effector protein to a monomer of Fe and then coupled through a disulfide bond to create a dimer, were not effective for large coagulation proteins such as FVIII. Thus, we have developed methods to create novel Fc fusion protein constructs in which a single (monomeric) effector molecule is attached to Fc (Dumont J.A., et al., BioDrugs 20(3):151-60 (2006)), (Dumont J.A., et al., Journal of aerosol medicine. 18(3):294-303 (2005)), (Bitonti, et al., Proc Natl Acad Sci USA. 10(26):9763 9768 (2004)). We have applied this approach to a number of proteins, including human rFIX (Peters, R.T, et al., Blood 115(10):2057-2064 (2010)), rFVIIa (Salas, J., et al., J Thromb Haemost. 9(s2):O-TU-026.doi: 10.1111/j.1538-78362011.04380_2x (2011)), and BDD rFVIII. Methods and Materials

[004581 Recombinant FVIII-Fe fusion protein (rFVIIIFc): The rFVIIIFc expression plasmid pBUDCE4.1 (Invitrogen) contained two expression cassettes. One expressed, under the control of CMV promoter, native human FVIII signal sequence followed by BDD FVIII (S743 to Q1638 fusion) directly linked to the Fc region of human IgG1 (amino acids D221 to K457, EU numbering) with no intervening sequence. The other used the EFla promoter to express the Fe region alone with a heterologous mouse IgB signal sequence. Human embryonic kidney 293 cells (HEK293H, Invitrogen) were transfected with this plasmid, and a stable clonal suspension cell line was generated that expressed rFVIIIFc. Protein was purified from defined cell culture harvest media using a three column purification process, including a FVIII-specific affinity purification step (McCue J. et al., J Chromatogr. A,, 1216(45): 7824 30 (2009)) followed by a combination of anion exchange and hydrophobic interaction chromatographicsteps.

[00459] Recombinant FVIII (rFVIII): Recombinant BDD FVIII (REFACTO* and XYNTHA*), and full length FVIII (ADVATE) were purchased from Novis Pharmaceuticals (Miami, FL) and reconstituted according to the manufacturer's instructions.

[00460J Animals: The hemophilia A (HemA) mice bearing a FVIII exon 16 knockout on a 129 x B6 background were obtained from Dr. H. Kazazian at the University of Pennsylvannia (Bi, L., et al., Nat Genet. 10(1):119-121 (1995)) and bred at Biogen Idec Hemophilia. Murine FeRn knockout (FeRn KO) and human FcRn transgenic (Tg32B) mice were derived from C57BL/6J mice and were obtained from Dr. Derry Roopenian of The Jackson Laboratory in Bar Harbor, ME. The genotypes for FcRn KO mice are mFeRn (-/-) and m2m (-/-), and for

Tg32B are mFcRn (-/-), mp2m (--),hFcRn (+/+), and hp2m (+/+). C57BL/6 mice were purchased from The Jackson Laboratories (Bar Harbor, ME). All animal activities were approved by the Institutional Animal Care Committees and performed in accordance with the "Guide to the Care and Use of Laboratory Animals."

[00461] Hemophilia A dogs were from the in-bred colony maintained at the Francis Owen Blood Research Laboratory at the University of North Carolina, Chapel Hill. These dogs have a severe hemophilic phenotype comparable to the severe form of the human disease (Graham, J.B. and Buckwalter, J.A., et al., The Journal of Exp. Med 90(2):97-111 (1949)), (Lozier, J.N., et al., Proc Natl Acad Sci U.S.A. 99(20):12991-12996 (2002)).

[00462] Pharmacokinetic (PK) Studies in mice: The PK of rFVIIIFc and rFVIII (XYNTHA*) was evaluated in HemA, C57BL/6, FcRn KO, and Tg32B mice after an intravenous dose of 125 IU/kg. Blood was collected from the vena cava in one-tenth volume of 4% sodium citrate at 5 minutes, and 4, 8, 16, 24, 32, 48, 54, and 72 hours post-dosing for rFVIIIFc and at 5 minutes, and 1, 4, 8, 16, 20, 24, 32, and 48 hours post-dosing for rFVIII (4 mice/time point/treatment). Plasma was snap frozen in an ethanol/dry ice bath and stored at -80°C until analysis for FVIII activity using a human FVIII-specific chromogenic assay (FVIII Coatest SP kit from DiaPharma [West Chester, OH]). The pharmacokinetic parameters were estimated by non-compartmental modeling using WINNONLIN1° version 5.2 (Pharsight, Mountain View, CA).

1004631 Efficacy studies in HemA mice: All efficacy studies were performed blinded. Acute efficacy was studied in the tail clip bleeding model. Male HemA mice (8-12 weeks old) were anesthetized with a cocktail of 50 mg/kg of Ketamine and 0.5 mg/kg of Dexmedetomidine. The tail was then immersed in 37C saline for 10 minutes to dilate the lateral vein followed by tail vein injection of rFVIIIFc, rFVIII (ADVATE), or vehicle. Five minutes later, the distal 1 cm of the tail was clipped and the shed blood was collected into 13 mL of warm saline for 30 minutes. The blood loss was quantified gravimetrically.

[00464] The prophylactic efficacy was studied in the tail vein transection (TVT) bleeding model as described previously (Pan, J. and Kim, J.Y., Blood 114(13):2802-2811 (2009)) except that HemA mice received a single intravenous administration of 12 lU/kg of rFVIIIFc, rFVIII (ADVATE*), or vehicle at 24 or 48 hours prior to the transection of a lateral tail vein. The dose of 12 IU/kg was identified from a prior dose response experiment with rFVIII in which 12 IU/kg achieved 50% protection of HemA mice from a TVT injury inflicted 24 hours post dosing (data not shown).

100465] Hemophilia A Dog Studies: In a single dose PK/PD study of rFVIIIFc, two naive hemophilia A dogs (M10 and Mll) received an intravenous dose of 125 IU/kg. Blood samples were collected pre-dosing and post-dosing at 5 and 30 min, and 1, 2, 4, 8, 24, 32, 48, 72, 96, 144, and 168 hours for whole blood clotting time (WBCT). Blood collections for FVIII activity (aPTT and chromogenic assay), rFVIIIFc antigen (ELISA), hematology, and blood chemistry included the time points listed above for WBCT as well as 15 minute and 3, 6, and 12 hours post-dosing.

100466] In the following sequential design study, rFVIII (REFACTO*) was administered intravenously at 114 IlU/kg for dog M12 and 120 IU/kg for dogM38. WBCT was measured until clotting times were > 20 minutes (the time consistent with FVIII:C < 1%), and samples were also collected at the specified time points for FVIII activity (aPTT and chromogenic assay), antigen (ELISA). and hematology tests. Then 125 IU/kg rFVIIIFc was administered intravenously to the same dogs and blood samples were collected for WBCT, aPTT, ELISA, hematology, and serum chemistry. Time points for WBCT included pre-dosing, and 5 and 30 minutes and 1, 2, 4, 8, 24, 32, 48, and 72 hours post-dosing of rFVIII and rFVIIIFc. Blood was also collected at 96, 120, 144, and 168 hours post-dosing with FVIIIFc. Blood collections for FVIII activity and antigens included the time points listed above for WBCT as well as 15 minutes and 3, 6, 12 hours after dosing. The WBCT and aPTT were performed as previously described (Herzog, et aL, Nat Med5 (1):56-63 (1999)).

[00467] FVIII chromogenic assays: FVIII activity in hemophilia A dog plasma was tested by an automated chromogenic assay on a Sysmex CA1500 instrument (Sysmex, IL) with reagents from Siemens Healthcare Diagnostics (Dallas, TX). The standard curve was generated with the 7th International Standard FVIII Concentrate (NIBSC code 99/678) spiked into human FVIII-depleted plasma (Stago, USA) at concentrations ranging from 1.5 - 0.016 IU/mL. 100468] FVIII activity in HemA mouse plasma was measured using the Coatest SP FVIII assay from Chromogenix (DiaPharma, Lexington, MA), following the manufacturer's instructions. The standard curve was generated using rFVIIIFc or rFVIII serially diluted from 100 mU/mL to 0.78 mU/mL in buffer containing naive HemA mouse plasma. To measure the human FVIII activity in C57BL/6, FcRn KO, and Tg32B mouse plasma, the infused rFVIIIFc or rFVIII in mouse plasma was first captured by human FVIII-specific mAb GMA8016 (Green Mountain Antibodies, VT) followed by the standard Coatest assay.

- ill -

[00469j rFVIII- and rFVIIIFc-specifc ELISA: rFVIII and rFVIIIFc antigen levels in hemophilia A dog plasma were measured by ELISA following the standard protocol. The FV111 Al domain-specific mAb GMA-8002 (Green Mountain Antibodies, Burlington, VT) was used as the capture antibody. HRP-conjugated polyclonal anti-FVIII Ab F8C-EIA-D (Affinity Biologicals) was used to detect rFVII HRP-conjugated donkey anti-human (F(ab) 2 ) 709-036-098 (Jackson Immunologicals) was used to detect rFVIIIFc. 1004701 SPR analysis of rFVIIFc-FcRn interactions: Surface plasmon resonance (SPR) experiments were performed with a Biacore T100 instrument. Research-grade CM5 sensor chips, buffers, and immobilization reagents were purchased from Biacore (GE Heathare, Piscataway, NJ). Single-chain human, canine, and murine FcRn preparations were immobilized using standard amine coupling on adjacent flow cells of a single chip at a density of approximately 370 resonance units (RU), followed by blocking with ethanolamine. The steady-state association of Fc-containing analytes (FVIIIFc and IgG) with immobilized FcRn of different species was evaluated by sequential injection of analytes at 16 concentrations (0.0625-2000 nM) in pH 6.0 running buffer (50 mM MES [4 morpholineethanesulfonic acid], 250 mM sodium chloride, 2 mM calcium chloride, 0.01% Tween 20 [polyethylene glycol sorbitan monolaurate]). Each cycle was performed in duplicate and comprised a 45 minutes association phase and a 15 minutes dissociation phase, both at a flow rate of 5pL/min, followed by regeneration with two 60 see injections of IM Tris-HCl at 25 L/min. After double reference-subtraction (blank flow cell and running buffer alone), binding responses recorded near the end of the association phase were plotted as a function of analyte concentration, and EC0 values (50% of Rmax) were derived by non linear regression analysis.

[00471] StatisticalAnalyses: Unpaired t-test, one-way ANOVA, Mann-Whitney test, Kruskal Wallis test with Dunn post-test, survival curves and associated log-rank test were performed in GraphPad Prism 5 (Graph-Pad Software Inc., La Jolla, CA). A 2-tailed P value less than 0.05 was considered statistically significant. Results

[00472] Recombinant FVIIIFcfusion protein (rFVIIHFc): rFVIIIFc is a recombinant fusion of human B-domain deleted FVIII with Fe from human IgG1, with no intervening linker sequence (FIG. 14), that was produced in well characterized HEK 293H cells. The rFVIIIFc is proteolytically cleaved intracellularly to yield an -90 kDa heavy chain and -130 kDa light chain-Fc that are bound together non-covalently through a metal bond interaction mediated by the Al and A3 domains of FVIII.

[00473] The average specific activity of rFVIIIFc from fourteen separate batches was 8460±699 IU/mg by the one stage clotting (aPTT) assay, and 9348+1353 IU/mg by the chromogenic assay, corresponding to 1861+154 and 2057+298 IU/nmol, respectively. The specific activity of rFVIIIFc is comparable to that of wild type human FVIII in plasma (1429 IU/nmol) (Butenas, S. and Mann, K.G., Biochemistry (Mosc). 67(1):3-12 (2002)). Thus the FVIII activity of rFVIIIFc is not affected by fusion of the C-terminus of human FVIII to the N-terminus of human Fe, and the results obtained with the aPTT and chromogenic assays are within approximately 10% of one another. 1004741 Binding of rFVIIFc to FcRn: The affinity of rFVIIIFc for single-chain mouse, canine, and human FeRn was evaluated using surface plasmon resonance. The rates of association and dissociation for the complex between rFVIIIFc and mouse FcRn were much slower than those for canine and human FeRn. Half-maximal binding (EC5 o) of rFVIIIFc to human FcRn was approximately 4-fold weaker than that to canine FcRn, and more than 20 fold weaker than that to mouse FcRn (TABLE 11). Similarly, human IgG1 also showed the highest affinity to murine FcRn, while binding affinity to canine FcRn was less compared to murine FcRn, but greater compared to human FcRn (TABLE 11).

TABLE 11. Surface Plasmon resonance analysis of murine, canine, and human FcRn with rFVIIIFc and human IgG1

Fc Sample FcRn FeRn Density (RU) EC50 (nM) t Rmax (RU) rFVIIIFc murine 370 < 1.5 581.4 rFVIIIFc canine 367 8.6 499.3 rFVIIIFc human 369 33.4 365.4 Human IgG1 murine 378 < 22.4 320.0 Human IgGI canine 367 196.3 282.2 Human IgG1 human 378 558.4 211.0

rFVIIIFc or IgGi were injected over a flow cell to which various FcRn molecules were chemically conjugated at approximately equal densities (-370 RU).

IEC 5 0 values (50% of Rm.) were the average derived from non-linear regression analysis of the binding response curves fitted to 16 analyte concentrations (0.0625-2000 nM) repeated in duplicate. IDue to the high affinities, the low binding curves at low analyte concentrations did not reach equilibrium under the normal operating conditions of the instrument.

[00475 FcRn-dependent improvement in pharmacokinetics of rFVIIIFc in mice: Interaction of Fc with FcRn is considered the underlying mechanism for extending half-life for IgG and Fc-fusion proteins. To confirm that this mechanism of action is also responsible for extending half-life of rFVIIIFc, we compared pharmacokinetic (PK) profiles of rFVIIIFc with rFVIII in FVIII-deficient (HemA) mice (FIG. 15A), normal (C57BL/6) mice (FIG. 15B), FcRn-deficient (FcRn KO) mice (FIG. 15C), and human FcRn transgenic (Tg32B) mice (FIG. 15D) following a single intravenous administration of 125 IU/kg.

[00476] The PK parameters (TABLE 12) were determined by the chromogenic measurement of the human FVIII activity in mouse plasma. The t/2 of rFVIIIFc was 1.8 - 2.2 fold longer than rFVIII in HemA mice (13.7 vs 7.6 hours) and normal mice (9.6 vs 4.3 hours). The ti/2 extension of rFVIIIFc relative to rFVIII was abolished in FcRn KO mice (6.4 vs 6.9 hours) and restored in human FcRn transgenic Tg32B mice (9.6 vs 4.1 hours). The results thus confirm that the interaction of rFVIIIFc with the FcRn receptor is responsible for its extended t 1/ 2 . Furthermore, consistent with the improved ti/ 2 , rFVIIIFc also showed a 1.6 2.4 fold longer MRT and a 1.2 - 1.8 fold increased systemic exposure (AUC) compared to rFVIII in FcRn-expressing (HemA, C57Bl/6 and Tg32B) mice but not in FcRn KO mice. TABLE 12. Summary of PK parameters for rFVIIIFc and rFVIII in different mouse strains

HemAt C57BL/6i FcRn KOt hFcRn Transgenic Mouse Strain (Tg32B)t rFVIIIFc rFVIII* rFVIIIFc* rFVIII* rFVIIIFc* rFVIII* rFVIIIFc* rFVIII*

2613.6 2710.4 2356.2 2000.1 2734.9 2458.4 3135.3 3137.0 (mIJ/mL) Half-life 13.7 7.6 9.6 4.3 6.4 6.9 9.6 4.1 (hr) MRT 17.6 11.0 9.8 5.4 6.3 8.5 12.8 5.4 (hr) /kg 68.2 49.2 67.5 50.8 49.3 51.6 64.1 49.6 (mL/kg) 6. CL 3.9 4.5 6.9 9.3 7.8 6.1 4.1 7.3 (mL/hr/kg) AUC (hr*mIU/mL 32332.4 28026.8 18089.1 13404.0 16087.2 20609.3 30534.5 17165.7

Cmax: maximum plasma FVIII activity post infusion; MRT: mean residence time; Vss: volume of distribution at steady-state;' CL: clearance;' AUC: area under the curve. iPK parameters of rFVIII and rFVIIIFc were compared only within the same mouse strain not across strains, because the same molecule can display different t/2 in different mouse strains. *The PK evaluation of each molecule used a cohort of 36 mice, sampled by terminal vena cava bleeding from 4 mice at each of the 9 time points. The group means at each time point were used for non-compartment modeling in WINNONLIN* to derive PK parameter estimates.

[00477 rFVIIIFc is fully active in treating acute bleeds in HemA mice: To evaluate the acute efficacy of rFVIIIFc in comparison to rFVIII, HemA mice (16-20 mice/group) were treated with escalating doses (24, 72, and 216 IU/kg) of rFVIIIFc or rFVIII and injured by tail clip 5 minutes post-dosing. In comparison to vehicle-treated mice (n=18) that had a median blood loss of 1 mL, both rFVIIIFc and rFVIII treatments resulted in significantly improved protection (P<0.05, Kruskal-Wallis test with Dunn post-test) (FIG. 16). The median blood loss progressively decreased with increasing doses, reaching maximum reduction to 0.23 mL at 72 IU/kg of rFVIIIFc, and 0.20 mL at 216 IU/kg of rFVIII. Overall, the blood loss was comparable in animals treated with equal doses of rFVIIIFc or rFVIII, indicating that both therapeutics are comparably active in resolving acute arterial bleeds.

[00478 Prolonged prophylactic efficacy of rFVIIIFc in HemA mice: To determine if prolonged PK lead to prolonged protection from injury, we compared the prophylactic efficacy of rFVIIIFc and rFVIII in HemA mice. Twenty-four hours after an intravenous dose of 12 IU/kg, one lateral tail vein in HemA mice was transected. Following injury, 49% of rFVIII-treated mice (n=39) survived, compared with 100% survival of rFVIIIFc-treated mice (n=19) (P<0.001, Log-Rank test) (FIG. 17A). To further demonstrate that rFVIIIFc sustains a longer duration of efficacy, HemA mice were injured 48 hours post-dosing with 12 IU/kg of rFVIIIFc. Nevertheless, 58% of rFVIIIFc-treated mice (n=40) survived, which is similar to that achieved in rFVIII-treated mice (49%) injured at 24 hours post dosing (FIG. 17A). Both

[Text continues on page 116] rFVIIIFc and rFVIII treatments are significantly better than the HemA vehicle-control group (n-30) in which only 3% of mice survived the injury (P<0.0001) (FIG. 17A). The improved and prolonged prophylactic efficacy of rFVIIIFc is also evident by the measurement of rebleeding post injury (FIG. 17B). Whereas 100% of vehicle-treated HemA mice rebled within 10 hours following tail vein transection, 87% of rFVIII-treated and 47% of rFVIIIFc treated mice rebled after the injury inflicted at 24 hours post dosing, respectively (P=0.002, rFVIIIFc vs rFVIII) (FIG. 17B). The rebleed profile for rFVIIIFc-treated mice injured at 48 hours is largely comparable to that for rFVIII-treated mice injured at 24 hours post dosing. In contrast, both the survival and rebleed profile for rFVIII-treated mice injured at 48 hours are indistinguishable from the profile for the vehicle-control group (data not shown). Therefore, the results indicate that rFVIIIFc protects HemA mice from tail vein injury twice as long as that achieved by the same dose of rFVIII.

[00479] Improved PK/PD of rFVIIFc in Hemophilia A dogs: The PK and pharmacodynamics (PD) of rFVIIIFc were also studied in hemophilia A dogs. Following an intravenous dose of 125 IU/kg of rFVIIIFc, the WBCT was immediately corrected to normal, which is in the range of 8 - 12 minutes in normal dogs (FIGs. 18A and B). The WBCT remained below 20 min, indicating FVIII activity >1%, through approximately 4 days in 3 out of 4 rFVIIIFc-treated dogs and 3 days in the remaining dog (FIG. 18A). In dog M12 treated with 114 IU/kg of rFVIII and dog M38 with 120 IU/kg of rFVIII, the WBCT was also corrected to normal immediately after dosing. However, the WBCT remained below 20 minutes for 2 days in M12 and 3 days in M38, approximately 1.5-2-fold shorter than that achieved by rFVIIIFc (FIG. 18B). Furthermore, both rFVIIIFc and rFVIII treatment also improved aPTT clotting time similarly at 5 minutes post dosing.

[004801 The PK of rFVIIIFc antigen (FIG. 19A) was determined by measuring the concentration of rFVIIIFc in plasma with a rFVIIIFc-specific ELISA that detects both the FVIII and Fc portions of the molecule. The tV of rFVIIIFc antigen is 15.7 + 1.7 hr (FIG. 19A), similar to the t, of rFVIIIFc activity (FIG. 19B), as measured by the chromogenic assay: 15.4 0.3 hr (TABLE 13). There is a good correlation between the FVIII activity and the rFVIIIFc antigen data, thereby demonstrating that rFVIIIFc protein was fully active in vivo.

TABLE 13. Summary of PK parameters for rFVIIIFc and rFVIII in hemophilia A dogs

PK by FVIII activity measurement AUC T1v 2 CL Vss Treatment Cmax (IU/mL) (hr-IU/mL) (hr) (mL/hr/kg) (mL/kg) rFVIIIFc' 2.0 0.54 25.9 6.47 15.4+0.3 5.1 1.4 86.4 14.0 rFVIIIT 2.0 18.2 7.4 6.5 64.0 PK by rFVIII and rFVIIIFc antigen measurement Cmax AUC T1v 2 CL Vss Treatment (ng/mL) (hr-ng/mL (hr) (mL/hr/kg) (mL/kg) rFVIIIFc' 210 ±33 2481 970 15.7 1.7 6.2+3.0 86.1 ±19.2 rFVIII* 211 1545 6.9 8.7 80.7

Results presented are Mean SD from 4 dogs. tResults presented are Mean. SD not reported since two dogs were utilized.

[004811 In two of the dogs (M12 and M38) that also received a single dose of rFVIII 72 hours prior to dosing with rFVIIIFe, the tv2 of rFVIII antigen was determined to be 6.9 hours and rFVIII activity 7.4 hours. Therefore, the plasma half-life of rFVIIIFc was approximately twice as long compared to that for rFVIII by both antigen and activity measurements.

[00482] In addition, platelet count and fibrinogen were assessed to serve as preliminary tests for thrombogenicity. After dosing with either rFVIIIFc or rFVIII, platelet numbers and plasma fibrinogen concentration did not change from pre-dose values (data not shown). Discussion

[00483] These studies have shown rFVIIIFe to be fully active in treating acute bleeds in HemA mice, in addition to retaining normal specific activity. Other studies, not reported here, have shown that rFVIIIFc is also fully functional in interacting with FIXa, FX, and phospholipids in forming the Xase complex (Peters et al., JI Thromb Haemost. DOI: 10.1111/jth.12076 (2012)). Furthermore, the binding affinity to von Willebrand Factor (VWF) was comparable between rFVIIIFc and rFVIII, with a Kd of approximately 1.4 and 0.8 nM for rFVIIIFc and rFVIII, respectively (Peters et al, Thromb Haemost. DOI: 10.1111/jth.12076 (2012)).

[00484] The activity of rFVIIIFc was not affected by fusion of the C-terminus of FVIII with the N-terminus of Fec since the Cl and C2 domains of FVIII were involved in phospholipid binding which is essential for the formation of prothrombinase complex on activated platelet surfaces (Foster, P.A., et al, Blood 75(10):1999-2004 (1990)). However, this finding was consistent with the observation that residues thought to bind phospholipids, e.g., K2092/F2093 in C1, M2199/F2200 and L225/L2252 in C2, all appear to form a surface that is distant from the C-terminal residues of FVIII (Shen, B.W., et al., Blood. (2007); Ngo, J.C., et al., Structure. 16(4):597-606 (2008)).

[00485] The half-life of rFVIIIFc was doubled only in mice expressing either endogenous murine or transgenic human FcRn, but not in FcRn KO mice (see FIG. 15 and TABLE 12), demonstrating that the mechanism of prolonging half-life of rFVIIIFc is mediated by FeRn. While it is known that both endothelial and hematopoietic cells contribute equally in recycling internalized IgG to the cell surface to facilitate the longevity of IgG and protection from degradation, (Borvak, J., et al., Int Immunol. 10(9):1289-1298 (1998)), (Akilesh, S., et al., J Immunol 179(7):4580-4588 (2007)) it is not known specifically which FcRn expressing cell type(s) are responsible for the uptake and recycling of rFVIIIFc. FcRn is broadly expressed in the vascular endothelium, epithelium of kidney, liver, spleen, as well as in bone marrow-derived APCs including macrophages (Borvak, J., et al., Int Immunol. 10(9):1289-1298 (1998)), (Akilesh, S., et al., J Immunol. 179(7):4580-4588 (2007)), (Yoshida, M., et al., Immunity. 20(6):769-783 (2004)). Since FVIII circulates largely (98 %) in complex with VWF (Lenting, P.J., et al., J Thromb Haemost. 5(7):1353-1360 (2007)), and both proteins were colocalized to the macrophages in liver and spleen when recombinant FVIII and VWF were co-injected into VWF-deficient mice (van Schooten, C.J., et al, Blood 112(5):1704-1712 (2008)), macrophages can play a role in rescue of rFVIIIFc from degradation and prolongation of half-life. However, the results can also be indicative of a previously unrecognized pathway for FVIII catabolism, and rescue of the protein permitted by Fc fusion.

[00486] Approaches in development to extend the half-life of clotting factors include pegylation (Rostin, J., et al., Bioconjug Chem. 11(3):387-396 (2000)), (Mei, B., et al., Blood 116(2):270-279 (2010)), glycopegylation (Moss, J., et al., J Thromb Haemost. 9(7):1368 1374 (2011)), (Negrier, C., et al., Blood. (2011)), and conjugation with albumin (Metzner, H.J., et al., Thromb Haemost. 102(4):634-644 (2009)), (Weimer, T., et al., Thromb Haemost. 99(4):659-667 (2008)). Regardless of the protein engineering utilized, the half-life of modified rFVIII variants appears to be maximally twice as long as wild-type FVIII in a variety of preclinical animal models (Liu, T., et al, Blood 112:511 (2008)), (Karpf, D.M., el al., 16(Suppl. S4):40 (2010)). Consistent results have been demonstrated in humans, e.g., rFVIIIFc was reported to improve half-life approximately 1.7-fold compared to ADVATE* in hemophilia A patients (Powell, J.S., et al., Blood (2012) prepublished online. DOI:10.1182/blood-2011-09-382846). This limitation of extending FVIII half-life appears to be related to VWF. In FVIII and VWF knockout mice, preliminary experiments observed a 5 fold increase in the half-life of rFVIIIFc compared to rFVIII (Liu, T et al., unpublished results). Similar findings were reported previously in VWF knockout mice utilizing Pegylated-FVIII (Mei, B., et al., Blood 116(2):270-279 (2010)). Taken together, these results indicate that VWF can be a limiting factor for further extending FVIII half-life.

[00487] Beyond extending half-life, rFVIIIFc provides additional benefits. One major challenge with FVIII replacement therapy is the development of neutralizing anti-FVIII antibodies (inhibitors). This occurs in 15-30% of previously untreated patients. rFVIIIFc has the potential to induce immune tolerance and thus prevent the development of neutralizing antibodies. It has been reported that retroviral vector-transduced B-cells, presenting FVIII domains as Ig fusion proteins, specifically prevent or decrease existing FVIII antibodies in HemA mice (Lei, T.C. and Scott, D.W., Blood 105(12):4865-4870 (2005)). It was also found that Fc contains regulatory T-cell epitopes capable of inducing Treg expansion and suppression of antigen-specific immune responses in vitro (De Groot, et al, Blood 112(8):3303-3311 (2008)). In addition, the FcRn-mediated transfer of maternal IgG and Fe fusion proteins across placenta to fetal circulation (Simister, N.E., Vaccine. 21(24):3365 3369 (2003)), Grubb, J.H., et al, Proc Natl Acad Sci USA 105(24):8375-8380 (2008)) could induce neonatal tolerance to rFVIIIFc while also providing needed protection in the newborn from bleeding during delivery.

[004881 In conclusion, we have demonstrated that rFVIIIFc, provides approximately 2-fold longer efficacy duration relative to rFVIII in protecting HemA mice from tail vein transection injury and improving WBCT in HemA dogs. The prolonged efficacy correlates well with a 2 fold extended t/ of rFVIIIF, a result of recycling of the Fc fusion protein via a specific and well established intracellular pathway.

Example 11 Immunogenicity of rFVIIIFc in Mice

[00489] 130 male Hemophilia A mice, age 7-9 weeks old at the beginning of the study, were randomized into 13 treatment groups based on age and Body weight (n=I0/group). Mice were treated with repeated intravenous dosing of either rFVIIIFc , rFVIII-mF, XYNTHA* or ADVATE* at 50, 100 and 250 IU/kg, a mixture of the three formulation buffers for FVIIIFc, XYNTHA* and ADVATE* was used for the vehicle control group. The IV administration times were day 0, day 7, day 14, day 21, day 35 post the first IV injection and blood samples were collected via Retro-orbital blood collection at day (-1), day 14, day 21, day 28 and day 42 post the first treatment (FIG. 20).

[00490] Immediately after blood collection, plasma samples were isolated through centrifugation and inactivated by 30 minute heat treatment at 560 C to insure the accurate measurement of anti-FVIII antibodies. The development of total anti-FVIII antibody (FIG. 21A, 21B and 21C), anti-FVIII neutralizing antibody (FIG. 23) and total anti-Fe antibody (FIG. 24) were investigated using the plasma samples.

[00491] When treated with 50 IU/kg FVIII, 28 days post the first injection, only 1 out of 10 mice in the rFVIIIFc treatment group, 2 out of 10 mice in the rFVIII-mFe treatment group developed detectable anti-FVIII antibody compared to 5 out of 10 mice and 7 out of 10 mice for the XYNTHA* and ADVATE* treated mice (FIG. 22C). At 100 IU/kg, the numbers of mice that had detectable anti-FVIII antibody at day 28 were 2, 5, 8 and 9 in the rFVIIIFc, rFVIII-mF, XYNTHA* and ADVATE* treatment group respectively (FIG. 22C). At 250 IU/kg, a supra- physiological dose, the numbers of mice thathad detectable anti-FVIII antibody at day28 were 10, 10, 7 and 7 in the rFVIIIFe, rFVIII-mFc, XYNTHA and ADVATE* treatment group respectively (FIG. 22C). Data corresponding to day 14, day 21, and day 42 is shown in FIGs. 22A, 22B, and 22D, respectively. 100492] In general, we observed a good correlation between the total and neutralizing antibodies to FVIII (R 2=0.7452), and the titers of both increased over time (FIG. 23). Within the therapeutic dose range (50 and 100 IU/kg), the number of mice that developed FVIII specific antibodies as well as the antibody titers in rFVIIIFc treatment groups were significantly lower compared to ADVATE* (p<0.05), and marginally lower versus XYNTHA®(p=0.05). The results indicate a potentially low immunogenicity of rFVIIIFc.

Example 12 Splenic Lymphocyte Response to rFVIIIFc Compared with Commercially Available rFVIII 1004931 The splenic lymphocyte response to recombinant Factor VIII (rFVIII) when linked to either human Fc (hFc, IgGI) or mouse Fc (mFc, IgG2a) was determined and compared with the splenic lymphocyte response to commercially available rFVIII [full-length FVIII (ADVATE*) and B-domain-deleted FVIII (XYNTHA®/REFACTO AF*)].

[00494] HemA mice were injected once weekly for six weeks with either 50 or 250 IU/kg of the tested molecules. On day 56, four mice from each group were euthanized and splenocytes were isolated (FIG. 25). One half of the splenocytes was used for determining the splenic immunogenicity profile by staining for intracellular cytokines, markers for regulatory T cells, and dendritic cells using flow cytometry (FACS) (FIG. 26). The other half of the cells were used for isolating RNA and carrying out pathway profiling using real time PCR based arrays. FIG. 27A shows a FACS dot plot profile of the isotype control. FIG. 27B shows a FACS dot plot profile of a sample containing splenocytes positive for both CD4 and TNF-a. Percentage of double positive cells were determined from dot plots from all the treatments and vehicle. Percent of double positive cells in FVIII treated mice was obtained by comparing with vehicle treated group.

[00495] Intracellular cytokine staining was performed by co-staining for the T-helper cell marker CD4 and cytokines such as IL-2 (FIG. 28), IL-4 (FIG. 30), and TNF-a (FIG. 29). IL-2 is a T-cell mitogen involved in T-cell proliferation, which is secreted by activated T-cells in response to FVIII in hemA mice. IL-4 has been identified as a cytokine secreted by activated T cells in response to FVIII in hemA mice. TNF-a is a pro-inflammatory cytokine responsible for higher antibody production in hemophilia patients. The fluorescence intensities of each of the staining were measured using flow cytometry.

[004961 Similarly, the proportion of tolerogenic and immunogenic dendritic cells was determined by surface staining and flow cytometry analysis of markers such as PD-Ll (CD274) (FIG. 32) and CD80 (FIG. 33). PD-LI is an inhibitory ligand that engages the PD-1 receptor on activated T-cells thereby blocking T-cell Receptor (TCR) mediated production of IL-2 and proliferation. Higher DC expression of PD-Ll is a critical factor that can inhibit immunogenicity and promote tolerance. CD80 is a surface marker usually seen on dendritic cells upon phagocytosing antigen and during maturation to present antigen to T cells. CD80 belongs to a panel of co-receptors in activating T cells for proliferation. More CD80 surface staining indirectly indicates better maturation and antigen presentation by dendritic cells.

[00497] In addition, the percentage of regulatory cells (Treg) in the spleen was assessed by co staining for CD4 and foxp3 (FIG. 34), a marker for these cells. Foxp3 is an intracellular marker of regulatory T cells. Foxp3+ T-cells are involved in the establishment, maintenance, and adoptive transfer of T-cell mediated peripheral tolerance.

[00498] The presence of cells expressing both CD4 and the Tim3 (FIG. 35) or CD279 (PD-1) (FIG. 36) makers was also assessed. Tim3 (T-cell immunoglobulin domain and mucin domain 3) as a negative regulator of T helper 1 (Thl)-cell responses. Tim3 is also expressed by innate immune cells and can promote a pro-inflammatory response. Tim3 inhibits Thl mediated auto- and alloimmune responses and acts via its ligand, galectin-9, to induce cell death in Thi but not Th2 cells. CD279 (PD-1) is a member of the extended CD28/CTLA-4 family of T cell regulators. PD-i is expressed on the surface of activated T cells, B cells, and macrophages suggesting that compared to CTLA-4, PD-1 more broadly negatively regulates immune responses.

[00499] Among the tested cytokines that are responsible for immunogenicity and inhibitor formation, in mice injected with the 50 IU/kg of rFVIII-hFc or rFVIII-mFe, there was a significant inhibition in the levels of IL-4 and TNF-a and the levels of IL-2 did not change compared to vehicle injected group. Conversely, the levels of these cytokines were higher in groups receiving 250 IU/kg of these molecules. Mice injected with 50 IU/kg of either XYNTHA* or ADVATE* did not exhibit any inhibition, whereas the 250 IU/kg group showed an increase in intracellular content of IL-2, IL-4, and TNF-cc. In addition, there was a higher percentage of foxp3 positive T cells in mice injected with 50 IU/kg of rFVIII-mFc compared to other treatments. Mice receiving 50 IU/kg of rFVIII-hFc and rFVIII-mFc had a higher percentage of splenic dendritic cells positive for PD-Ll (CD274), an inhibitory signal for T-cell activation and proliferation. These groups also had a higher percentage of immature dendritic cells as illustrated by a decrease in CD80 staining.

[00500] These results indicated that rFVIIIFc at 50 IU/kg in hemA mice exhibited lower immunogenicity than commercially available rFVIII [full-length FVIII (ADVATE*) and B domain-deleted FVIII (XYNTHA/REFACTO AF)]. Accordingly, rFVIIIFc can promote lower antibody production and induce immune tolerance.

Example 13 Biodistribution and clearance of rFVIIIFc in mice

[005011 Recombinant fusion of a single FVIII molecule to the constant region of IgG1 Fc (rFVIIIFc) has been shown to decrease clearance compared to rFVIII in an FcRn dependent manner (Powell et al., 2012 Blood), using a natural pathway that recirculates antibodies into the blood stream. In addition, as disclosed above, a phase 1/2a clinical trial in hemophilia A subjects demonstrated that rFVIIIFc has a 1.5 to 1.7-fold longer half-life than recombinant full length FVIII (ADVATE*). Accordingly, a study was conducted (i) to identify the cell types and organs that contribute to the protection of rFVIIIFc and (ii) to assess the relative contributions of the FVII and Fe domains to the biodistribution and clearance of rFVIIIFc in mice.

[005021 The clearance of rFVIIIFc to rFVIII was compared in genetically engineered KO mouse models deficient in either FVIII (HemA) or von Willebrand Factor (VWF). Intravenously dosed clodronate-containing lipid vesicles were used to deplete Kupffer cells and monocytes/macrophages in these mouse models. The effectiveness of depletion was quantitated by immunohistochemistry and FACS analysis. Pharmacokinetic analysis was performed with a FVIII-specific Coatest assay following intravenous injection of rFVIIIFc or rFVIII.

[00503] Kupffer cell depletion in HemA mice increased rFVIIIFc clearance. Furthermore, in the absence of VWF (HemA/VWF double knockout mice), the depletion of Kupffer cells and macrophages increased the clearance of rFVIIIFc to levels similar to that of rFVIII, indicating that these cells are responsible for the majority of the difference in clearance between rFVIII and rFVIIIFc in this model.

[00504] These studies suggest that Kupffer cells can contribute to the FcRn-mediated recycling of rFVIIIFc. Studies using bone marrow transplants with FcRn KO mice are in progress to verify this mechanism. These studies, combined with in vitro cellular uptake experiments will attempt to distinguish the contribution of Kupffer cells from other FcRn expressing cell types, including endothelial cells.

Example 14 Cell-Mediated Immune Response to rFVHIFc in Hemophilia A Mice

[00505] The objective of the present study was to identify cell-mediated immune responses to recombinant FVIII, which is of interest in designing better therapeutic management of hemophilia A. Thus, we investigated the splenic lymphocyte response to recombinant FVIII (rFVIII) when linked to human Fc (rFVIIIFc; IgGI) in comparison with commercially available full-length rFVIII (ADVATE*) and B-domain-deleted rFVIII (XYNTHA*/REFACTO AFl). HenA mice were injected with 4 weekly followed by 2 every other week doses of 50 100 or 250 IU/kg. At the end of 8 weeks, mice from each group were euthanized and their splenic leukocyte immunogenicity profile was determined by testing for intracellular cytokines, markers for regulatory T cells and dendritic cells using flow cytometry and RNA profiling. In mice injected with the 50 lU/kg of rFVIIIFc, there was a significant inhibition in the levels of IL-2, IL-4 and TNF- (cytokines that promote immunogenicity). The levels of these cytokines were higher in mice receiving 250 IU/kg of this molecule. Mice injected with 50 IU/kg of either XYNTHA* or ADVATE* did not exhibit any inhibition, whereas the 250 IU/kg group showed an increase in intracellular content of IL-2, IL-4, and TNF-u. In addition, there was a higher percentage of foxp3 positive T cells in mice injected with 50 and 100 IU/kg of rFVIIIFc compared to other treatments. Mice receiving 50 and 100 IU/kg of rFVIIIFc had a higher percentage of splenic dendritic cells positive for PD-Li (CD279), an inhibitory signal for T-cell activation and proliferation. These groups also had a higher percentage of immature dendritic cells as illustrated by a decrease in CD80 staining. Thus, both the 50 and 100 IU/kg doses of rFVIIIFc showed lower immunogenicity and antibody production in this model.

Introduction

[005061 Development of inhibitors to FVIII is recognized as a serious complication in the management of hemophilia A. The incidence of inhibitor formation is estimated to range from 20% to 30% in all hemophilia A to 30 - 40 % in severe disease. (Green, Haemophilia 17:831-838 (2011); Eckhardt el al J. Thromb. Haemost. 9:1948-58 (2011)). Inhibitor positive disease is currently managed by immune tolerance induction involving the frequent high-dose administration of FVIII. Mechanisms of inhibitor formation in patients are largely unknown and depend on a multitude of risk factors and cells and molecules of the immune system.

[005071 Inhibitor development in hemophilia involves a complex interplay of multiple cell types, surface molecules and secreted proteins of the immune system including, T lymphocytes, B-lymphocytes, antigen presenting cells (APC; dendritic cells and macrophages), cytokines, and regulatory components of these cell types. Antibody production by B-cells depends on optimal help from T-cells, which are activated by antigen presentation from APC.

[005081 Tolerance to injected therapeutic peptides and proteins including recombinant FVIII is mediated by a class of T-cells called regulatory T-cells (Treg) (Cao et al, J. Thromb. Haemost. 7(S1):88-91 (2009)). Several key molecules have been identified that correlate with inhibitor formation in hemophilia patients. These include the pro-inflammatory cytokine TNF-a, the anti-inflammatory cytokine interleukin (IL)-10, and the Treg marker CTLA4, to name a few (Astermark et al., J. Thromb. Haemost. 5:263-5 (2007); Pavlova et al. J. Thromb. Haemost. 7:2006-15 (2009)).

[005091 In this study, we investigated the splenic lymphocyte response to recombinant factor VIII Fe fusion protein (rFVIIIFc) compared with commercially available full-length rFVIII (fl-rFVIII; ADVATE*) and B-domain deleted rFVIII (BDD-rFVIII; XYNTHA*/REFACTO AF*). Materials and Methods

[00510] Materials: Factor FVIII-deficient mice (Bi et aL Nat Genet. 10:119-21 (1995)) were originally acquired from Dr. Kazazian (University of Pennsylvania, Philadelphia, PA) and maintained as breeding colonies either at Biogen Idec Hemophilia or Charles River Laboratory.

[005111 Antibodies used for staining and FACS analysis were obtained either from BD Biosciences (Franklin Lakes, NJ, USA) or eBioscience (San Diego, CA, USA). Antibodies used were directed against murine surface markers such as CD4 (T-helper cells), CDI1c and CD80 (dendritic cells), PD-1, PD-L1, CD25 (Treg cells), intracellular cytokines (IL-2, IL-4, TNF-a), and transcription factors (Foxp3).

[00512] Immunogenicity Study Design: Three treatment groups received intravenous doses of 50, 100, or 250 IU/kg, which were administered on days (FIG. 37). Each treatment and dose level were administered to 10 mice. Animals were euthanised on day 56 by C02 inhalation and spleens were dissected in sterile PBS.

[00513J Splenocytes were separated using the mouse spleen dissociation kit and a gentle MACS dissociator (Miltenyi Biotec, Cologne, Germany). Single cell suspensions of splenocytes were either fixed in 3% formalin for FACS staining or stored in dissociation buffer for RNA isolation (Roche).

[005141 Assessments: Anti-FVIII antibodies were determined using an in-house developed ELISA. Briefly, FVIII was coated on 96-well plates and used to capture antibodies from mouse plasma collected at specific time points. FVIII-specific antibodies were detected using a secondary anti-mouse IgG antibody. T-cell response profiling was conducted on isolated mouse splenocytes (FIG. 38). Lymphocytes and dendritic cells in spleen were stained for surface and intracellular targets.

[005151 For surface staining, 1 x 106 total splenocytes were incubated with antibodies at appropriate concentrations. For intracellular staining, cells were permeabilised with BD Fix

Perm solution (BD Biosciences) followed by incubation with antibodies to cytokines in the same buffer. Foxp3 staining was carried out using antibody in Foxp3 staining buffer (BD Biosciences). Fluorescence intensity was recorded using a BD FACS Canto II and analysis performed using FLOWJO* software.

[00516] Lymphocytes were costained with CD4 and intracellular markers IL-2, TNF-a, and IL-4. Treg cells were stained for surface markers CD4 and CD25 followed by intracellular Foxp3. Splenocytes were stained for CD11e and PD-Li (dendritic cells) or CD4 and PD-1 (CD4+ T-cells) to identify cells involved in the PD-L1-PD-1 pathway. Total RNA was isolated (Roche) and reverse transcribed to cDNA (Qiagen, Hilden, Germany). Primers for TGF-p, IL-10, IL-12a, and EBI-3 were purchased from IDT technologies. SYBR green based real-time polymerase chain reaction (PCR) was carried out using Quantitect system (Qiagen) using an ABI 7900 Fast Block real-time PCR machine. Data were analysed using the 7500 software version 2.0.5. Results 1005171 Total anti-FVIII Antibody Levels on Day 42: Total anti-FVIII IgG levels on day 42 were assayed from plasma of haemophilia A mice injected with 50, 100 or 250 IU/kg of rFVIIIF, BDD-FVIII (XYNTHA*) or full length FVIII (fl-rFVIII) (ADVATE*) using ELISA. At both 50 and 100 IU/kg the rFVIIIFc group had significantly lower antibody levels compared to BDD-rFVIII and full length fl-FVIII, which indicated lower antigenicity to FVIII imparted by rFVIIIFc injections. At 250 IU/kg the groups were not significantly different from each other and had high antibody levels (FIG. 39).

[005181 Intracellular Cytokines (IL-2 and TNF-a) in CD4+ Cells: Mice receiving 50 U/kg in each-treatment group were nonresponders based on antibody levels to FVIII, whereas the mice receiving 250 IU/kg in each treatment group were responders with the highest antibody levels (data not shown). rFVIIIFc at 50 and 100 IU/kg doses (FIG. 40A and FIG. 40B) lowered the percentage of IL-2 and TNF-a positive CD4+ cells, which indicated lower immunogenicity whereas BDD rFVIII and full-length rFVIII showed higher cytokine positive cells. All 3 treatments at the 250 IU/kg dose (FIG. 40C) elevated the percentage of cytokine positive cells, which indicated higher immunogenicity at this dose. Similar results were obtained for IL-4. 1005191 CD4/CD25/Foxp3 Triple Positive Cells (Markers for Treg Cells): At the 100 IU/kg dose, the percentage increase of Treg cells over vehicle were significantly higher in the rFVIIIFc group (P<0.05) compared with BDD-rFVIII and fl-rFVIII groups (FIG. 41). Similar results were obtained for the 50 IU/kg of rFVIIIFc indicating that both the 50 and 100 IU/kg rFVIIIFc treatments can promote predominance of Treg cells and suppress immune responses to FVIIL

[00520] Real-Time Polymerase Chain Reactionfor Tolerance-Related Cytokines: Real Time PCR analysis for immuno tolerance related cytokines namely TGF-D (FIG. 42A), IL-10 (FIG. 42B) and IL-35 (IL-12a and EBI-3 subunits, shown respectively in FIGs. 42C and 42D) was carried out using RNA isolated from mice belonging to the 100 IU/kg treatment group. mRNA levels were upregulated for the tested cytokines in the rFVIIIFc group (P<0.05) compared with the other treatments at 100 IU/kg, which indicated the presence of splenocytes actively expressing tolerogenic cytokines thereby promoting an immunosuppressive microenvironment. mRNA expression immunotolerance markers Foxp3 (FIG. 42E), CD25 (FIG. 42F), CTLA-4 (FIG. 42G), and Indoleamine 2,3-dioxygenase (IDO-1) (FIG. 42H) were higher in total splenocytes from 100 IU/kg group

100521] FACS Analysis of PD-Li-PD-1 Pathway: PD-Li (CD274) on dendritic cells engage PD-1 (CD279) on T-cells to promote inhibitory pathways that suppress T-cell activation and proliferation, thereby leading to suppression of immune responses. Splenocytes from the 100 IU/kg group were stained for either surface CDl li and PD-Ll (FIG. 43A), or CD4 and PD-1 (FIG. 43B). At the 100 IU/kg dose, the percentage of dendritic cells positive for PD-Li and the percentage of T-cells positive for PD-1 were higher in the animals receiving rFVIIIFc (P<-.05) compared with those receiving BDD-FVIII and fl-rFVIII. This indicated positive regulation of immunosuppressive pathways at both dendritic cells and T-cells by rFVIIIFc. Discussion 1005221 The experimental results shown above indicated that rFVIIIFc at doses of 50 and 100 IU/kg had low immunogenicity and promoted tolerance to FVIII, as demonstrated by: (a) A lower level of pro-immunogenic cytokines (IL-2 and TNF-c) in CD4+ T-cells compared with other FVIII molecules; (b) An up-regulation of regulatory T-cells and markers (Foxp3, CD25, PD-1, CTLA4) that are responsible for immune tolerance in rFVIIIFc-injected mice. The significance of Foxp3+ Treg and the role of CTLA4 in promoting tolerance to FVIII in haemophilia have been previously described (Cao et al., J. Thromb. Haemost. 7(S1):88-91 (2009); Astermark el al J. Thromb. Haemost. 5:263-5 (2007)).

(c) A higher level of tolerogenic cytokines (11-10, TGF-P, IL-35) in splenocytes of mice injected with rFVIII. These markers have been shown in several studies to be key immunoregulatory cytokines and major determinants of immuno-tolerance (Bi el al., Nat. Genet. 10:119-21 (1995)). (d) An elevation of tolerogenic dendritic cell population (PD-Li, IDO-1, and decreased CD80) in mice following injection with rFVIIIFc. Concurrently, rFVIIIFc-treated mice also showed lower or no antibodies to FVIII at doses of 50 and 100 IU/kg (Liu et al, WFH Abstract #FB-WE-04.2-5 (2012)) and resisted challenge with 250 lU/kg once tolerised wuth 50 IU/kg of rFVIIIFc.

[00523] Conclusions: rFVIIIF-treated mice showed lower or no antibodies to FVIII at doses of 50 and 100 U/kg compared with traditional FVIII therapies. Based on T-cell and dendritic cell studies in mice, rFVIIIFc was found to be less immunogenic than traditional FVIII therapies and promoted tolerogenic pathways. rFVIIIFc upregulated key immunoregulatory cytokines in splenocytes of haemophilia A mice that are indicative of immunotolerance. Taken together these findings indicate the existence of a tolerogenic microenvironment in the spleen of mice injected with rFVIIIFc at low doses (50 and 100 IU/kg).

[00524] These studies have demonstrated for the first time that rFVIIIFc activates dendritic cell signaling, which is a crucial determinant of immunotolerance. These findings indicate the existence of functional immune tolerance to FVIII imparted by rFVIIIFc in haemophilia A mice.

Example 15 Evaluation of Antibody Responses to rFVIIIFc Compared to XYNTHA@ and ADVATE@ In Hemophilia A mice

[00525] Development of inhibitory antibodies to replacement FVIII occurs in 20-30% of previously untreated patients, being the most severe complication of hemophilia treatment. Immune tolerance induction (ITI), which entails frequent administration of FVIII, is currently used to treat patients who develop inhibitors. A subset of these patients, however, do not respond to ITI. See, e.g., Green, Haemophilia 17:831-838 (2011); Eckhardt et al. J. Thromb. Haemost. 9:1948-58 (2011); Cao et al, J. Thromb. Haemost. 7(S1):88-91 (2009). Recombinant FVIIIFc has a half-life approximately 1.6-fold longer than the half-life of rFVIII and it is currently in phase 2/3 clinical development. The experiments disclosed herein assess the immunogenicity and immune tolerance properties of rFVIIIFc compared with other rFVIII replacement proteins in hemophilia A (HemA) mice.

[00526] (a) Immunogenicity Comparison for rFVIIFc, XYNTHA* and ADVATE* in HemA Mice: Antibody Response: Four groups of HemA mice, with 9-12 HemA mice per group, were treated with 50 IU/kg, 100 IU/kg and 250 IU/kg doses of rFVIIIFe, XYNTHA*, ADVATE*, and vehicle control. Doses were administered at day 0, day 7, day 14, day 21, and day 35. Blood was drawn at day 0, day 14, day 21, day 28 and day 42 (FIG. 44).

[005271 rFVIIIFc induced a significantly lower antibody response at 50 IU/kg (FIG. 45) and at 100 IU/kg (FIG. 46) compared to XYNTHA* and ADVATE. However, all FVIII proteins showed similar antibody response at 250 IU/kg (FIG. 47). Neutralizing antibody titers correlated with total binding antibody levels (FIG. 48).

[00528] (b) Immunogenicity Comparison for rFVIIIFc, XYNTHA* and ADVATE* in HemA Mice: T-CeH Response Profiling: For T-cell response profiling in splenocytes, an additional dose of rFVIIIFc, XYNTHA* ADVATE*, and vehicle control was administered at day 53. Spleens were collected at day 56 (FIG. 49). The results indicated that rFVIIIFc promotes predominance of CD4/CD25/Foxp3-positive Treg Cells (FIG. 50, right panel).

100529] Summary: Administration of rFVIIIFc resulted in significantly lower antibody response compared with XYNTHA* and ADVATE at doses of 50 and 100 IU/kg. The tolerogenic profile following 50 and 100 IU/kg doses of rFVIIIFc indicated that rFVIIIFc can promote predominance of Treg cells and suppress immune response to FVIII

[00530] (c) rFVIH Immune Tolerization Study: To test whether repeated administration of rFVIIIFc can induce functional tolerance in vivo the following dosing regimen was adopted. HemA mice (8-10 wk old) were injected with 50 IU/kg of rFVIIIFc or vehicle every week for 4 weeks (on days 0, 7, 14, 21) followed by one injection on day 35. Starting day 49 these mice were challenged with weekly 250 IU/kg of rFVIIIFc to determine if the animals can tolerate high doses of rFVIIIFc. The challenge doses were administered on days 0, 7, 14, and 21 starting day 49 of the study (see FIG. 51). Blood samples were collected at specified time points to check for anti-FVIII antibodies using ELISA. As shown in FIG. 52, repeated dosing of rFVIIIFc led to statistically significant reduction in antibodies to rFVIIIFc when challenged at high doses of 250 IU/kg, while animals receiving vehicle had higher levels of antibodies upon challenge. This clearly indicates that repeated administration of rFVIIIFc at 50 IU/kg based on the dosing scheme followed can induce tolerance to higher doses of rFVIIIFc (250 IU/kg).

[00531] Conclusion: At therapeutic doses, rFVIIIFc was found to be (1) less immunogenic compared with XYNTHA* and ADVATE*, and (2) capable of inducing immune tolerance to FVIII in HemA mice. 1005321 Currently, its being determined whether even lower doses of rFVIIIFc can lead to immune tolerization to higher doses of rFVIIIFc. In the present study, lower doses of rFVIIIFc, namely 25 IU/kg and 10 IU/kg, are being used during the tolerance induction phase. This will be followed by challenging with 250 IU/kg of rFVIIIFc and measurement of antibodies to FVIII as carried out for the previous study.

Example 16 Clearance Pathways of rFVIIIFc in Haemophilia A Mice

[00533] Long-lasting recombinant coagulation FVIII Fc fusion protein (rFVIIIFc) is currently in phase 3 clinical study for episodic and prophylactic treatment of individuals with haemophilia A. Compared with recombinant full-length FVIII (ADVATE*, Baxter Healthcare Corporation), rFVIIIFc has a 1.7-fold extended half-life and significantly decreased clearance in patients with haemophilia A. See, Powell et al., Blood 119:3031 7(2012). This improved pharmacokinetic (PK) profile is mediated through the interaction of Fc with neonatal Fe receptor (FeRn). See Dumont et al, Blood 119:3024-30 (2012). rFVIIIFc is comprised of a single B-domain deleted human coagulation FVIII attached directly to the Fe domain of human immunoglobulin G1, which is naturally recycled upon cellular uptake (endocytosis or pinocytosis) through interaction with FcRn (FIG. 53). Monocytic cells (macrophages and dendritic cells), including liver resident macrophages (Kupffer cells), are implicated in the clearance of von Willebrand factor (VWF) and FVIII (FIG. 54). See van Schooten et al., Blood 112(5):1704-12 (2008). 1005341 In order to elucidate the cell types involved in rFVIIIFc uptake, clearance, and FcRn mediated recycling, we studied the impact of macrophage and Kupffer cell depletion on the clearance of rFVIIIFc in genetically engineered mouse models. Materials and Methods

[00535J The clearance of rFVIIIFc and rFVIII (BDD) was compared in 3 knockout (KO) mouse models: (1) haemophilia A (FVIII KO), deficient in FVIII; (2) double KO (DKO) of FVIII and VWF, lacking expression of both FVIII and VWF; and (3) FcRn-KO, lacking expression of the FcRn. In all 3 models, macrophage and Kupffer cells were depleted with CLODROSOME@ (Encapsula NanoSciences, Inc), which is a toxic ATP analogue

(clodronate) encapsulated in liposomes that is specifically phagocytosed by macrophages and triggers apoptosis. See van Rooijen & Hendrikx, Methods Mol. Biol. 605:189-203 (2010). 100536] Control mice in each group were treated with ENCAPOSOME* nontoxic liposomes (FIG. 55). A single intravenous dose of either FVIII or rFVIIIFc (125 or 250 IU/kg) was injected 24 hours after treatment with liposomes. Blood samples were collected by retroorbital or vena cava blood collection at specified time points (4 samples per time point).

[005371 Human FVIII activity in plasma samples was then measured by a FVIII chromogenic assay, and PK parameters were estimated with WINNONLIN* software (Pharsight Corp.) using a noncompartmental analysis model.

[005381 Kupffer cell and macrophage depletion was evaluated by immunohistochemical staining, and was quantified using Visiopharm software (Hoersholm, Denmark) or by reverse transcrition polymerase chain reaction (RT-PCR). Results

[005391 (a) Depletions of macrophages and Kupffer cells: FIG. 56 shows a representative staining of liver sections with an antibody to Iba-1, a specific macrophage marker, 24 hours after control ENCAPSOME* (A, A') or CLODROSOME* (B. B') treatment of haemophilia A mice. A' and B' show the quantification masks highlighting the stained Kupffer cells (azure), total tissue area (navy blue), and empty areas (grey). Similar depletion profiles were obtained in other mouse strains. Quantitative analysis of positively stained areas showed that >90% of liver Kupffer cells were depleted and remained low for >3 days post CLODROSOME* treatment (n=4) compared with control ENCAPSOME*-treated animals (FIG. 57). Circulating monocytic cells were also reduced by >50% within 24 hours of CLODROSOME* treatment, assessed by flow cytometric analysis of blood cells stained with a labeled antibody to F4/80+ (n=4). Within 48 hours, depleted blood cells recovered (FIG. 57). Consistent with the observed depletion of macrophages in the liver, RT-PCR analysis of the expression of the macrophage marker epidermal growth factor module-containing mucin like receptor 1 (Emrl) (F4/80) showed that CLODROSOME treatment decreased Emrl mRNA expression >95% in liver and lung in haemophilia A mice (FIG. 58). Emrl is the designation used for the human protein. The mouse homolog is known as F4/80. Emrl is a transmembrane protein present on the cell-surface of mature macrophages.

[00540J (b) Clearance of FVII in mouse models: In contrast to previously reported results that suggest Kupffer cells play a role in the clearance of both FVIII and VWF (van Schooten CJ, et al. Blood. 112(5):1704-12 (2008)), Kupffer cell depletion did not cause the expected decrease in FVIII clearance in haemophilia A mice (FIG. 59). To the contrary, depletion of Kupffer cells in haemophilia A mice significantly increased the clearance of rFVIIIFc (FIG. 59). Similarly, in DKO mice, Kupffer cell depletion did not decrease FVIII clearance and significantly increased clearance of rFVIIIFc (FIG. 60). In FcRn-KO mice, Kupffer cell depletion did not affect the clearance of FVIII or rFVIIIFc (FIG. 61).

[00541] Conclusions: Macrophage and Kupffer cell depletion in haemophilia A and DKO mice (deficient in FVIII and VWF) increased rFVIIIFc clearance, but did not decrease FVIII clearance, indicating that macrophage and Kupffer cells can account for much of the difference in clearance between FVIII and rFVIIIFc in these mice. In the absence of both VWF and FVIII (DKO mice), depletion of macrophages and Kupffer cells increased the clearance of rFVIIIFc to levels approaching that of FVIII.

[00542] The lack of effect of Kupffer cells depletion on FVIII clearance in either haemophilia A or DKO mice contrasted with previously published studies. See van Rooijen & Hendrikx, Methods Mol. Biol. 605:189-203 (2010). Macrophage and Kupffer cell depletion in FcRn KO mice did not result in significant clearance differences between FVIII and rFVIIIFc, indicating a potential role of FcRn in macrophage-mediated recycling of rFVIIIFc.

[00543] Together, these studies indicated that rFVIIIFc was protected in a macrophage- and/or Kupffer cell- dependent manner and that a natural pathway in these cells can contribute to FcRn-mediated recycling of rFVIIIFc.

Example 17 Immune Tolerization to Pre-Existing Antibodies to Factor VIII

[00544] It is highly likely that patients that receive rFVIIIFc were previously on a different rFVIII therapy. About 30 % of hemophilia A patients develop antibodies to FVIII, which is a major problem in current FVIII replacement therapy. Currently, the only approved approach to tolerize patients having FVIII inhibitors is to dose them with high doses of FVIII for an indefinite period of time, i.e., until the patient is tolerized. Thus, it is of interest to determine if low dose rFVIIIFc can reverse the immunogenicity of FVIII by inhibiting T and B cell signaling pathways and skewing towards tolerance.

[00545] In this study, hemA mice (8-10 wks old) will be injected weekly once for 5 weeks with either BDD-FVIII (XYNTHA*) or fl-FVIII (ADVATE*) at 50 IU/kg. Levels of anti FVIII antibodies will be determined in blood samples drawn from these animals on specified days as indicated in the scheme. Following the induction of inhibitory antibodies, animals will be switched to injections with 50 IU/kg of rFVIIIFc weekly for 5 weeks. This will be the tolerization phase. Levels of anti-FVIII antibodies will be determined again from blood samples drawn as indicated. Following the tolerization phase, animals will be injected again with XYNTHA* or ADVATE* at 50 IU/kg for another four injections and blood samples drawn will be tested for anti-FVIII antibodies. In the event of successful tolerization, the second challenge with XYNTHA* or ADVATE* should not produce any antibodies to FVIII which may indicate the capability of rFVIIIFc to induce immunotolerance to FVIIL.

Example 18 T-cell Adoptive Immunotherapy and Transfer of Immune Tolerance

[00546J Regulatory T-cells (Tregs) are the master players in the induction and maintenance of peripheral tolerance to FVIII and other peptide therapeutics. One of the key studies used in determining the presence of functional immune tolerance is by transferring Treg cells isolated from tolerized animals to recipients, which are then challenged with higher doses of FVIII. The presence of functional transfer of tolerance is evidenced by lack of or lesser antibody production in the challenged recipient mice. 1005471 In this study, hemA mice (8-10 weeks old) will be tolerized by injecting 50 IU/kg of rFVIIIFc or vehicle every week for 4 weeks (on days 0, 7, 14, 21) followed by two injections on days 35 and 53. Plasma samples will be collected for determination of anti-FVIII antibody levels using ELISA. On day 56, mice with no antibodies will be euthanized and their spleens will be isolated. Splenocytes from these mice will be harvested and made into single cell suspensions using the splenocyte isolation kit (Miltenyi Biotec). Tregs from total splenocytes will be isolated using the CD4 CD25 murine Treg magnetic bead based isolation kit (Miltenyi Biotech . Isolated Tregs will be enumerated using a cell counter. An aliquot of the cell suspension will be fixed using 3 % formalin for phenotypic analysis by FACS to determine the purity of the isolate. Tregs will then be injected into recipient mice on day 0 at 1 x 106 cells/mouse in 200 pL saline by IV.

[005481 Starting day 1 animals will be challenged with 250 lU/kg of rFVIIIFc followed by repeated injections weekly once on days 8, 15, 22, and 29. Blood samples will be collected on days 15, 22, 29 and 36 for plasma anti-FVIII antibody analysis using ELISA. In the event of successful transfer of tolerance, mice receiving Tregs from rFVIIIFc tolerized animals will not develop antibodies to FVIII whereas, mice injected with Tregs isolated from vehicle injected donor mice show the presence of anti-FVIII antibodies.

Example 19 Identification of Mechanisms of Immune Tolerance Induction by rFVIIIFc: Studies with Dendritic Cells and Macrophages

[00549] One of the key features that distinguishes rFVIIIFc from other FVIII drug products is the presence of the Fc moiety which is a naturally occurring constituent. This could be one responsible factor that suppresses the immune response and drives an immune tolerance reaction. Fe molecules present in IgG and in rFVIIIFc are capable of interacting with the classical IgG Fc receptors and FcRn. Among the Fc receptors, the subtype FcyRlIb (FcyR2b) is an inhibitory receptor and delivers suppressive signals that curb the activation of cell types harboring that receptor. Fe receptors including FcRn are chiefly localized in antigen presenting cells (APC - dendritic cells, macrophages and B-cells), but not in T-cells. Therefore, it is possible that the rFVIIIFc could engage FcR2b and/or FcRn in these cells to skew towards a tolerogenic phenotype.

[00550] To ascertain whether rFVIIIFc engages FcR2b and/or FcRn in dendritic cells and macrophages to develop a tolerogenic phenotype, the following experiments will be conducted: 1. Identification of markers at the mRNA and cell surface levels (protein) in murine macrophage cell lines, splenic macrophages and splenic dendritic cells that are regulated by rFVIIIFc in comparison with FVIII alone or a mutant of FVIIIFc (N297A) which does not interact with Fe receptors. 2. Overexpression and knockdown of FcgR2b or FcRn in RAW 264.7 murine macrophage cell line and investigating the effects of rFVIIIFc on these receptors by studying downstream targets 3. Identification of the possible involvement of other pathways of importance such as TLR mediated signaling in the APC response to rFVIIIFc

Example 20 Alternate Routes for Induction of Tolerance

[005511 Immune tolerance to rFVIIIFc may also be achieved via mucosal route. Two possible sites for inducing tolerance are the gastrointestinal mucosa and the respiratory mucosa. Oral tolerance to rFVIIIFc can be induced by either feeding animals or using oral gavages to deliver the molecule to the intestinal mucosa. The gut mucosa has specialized secondary lymphoid organs namely the Peyer's patches which contains antigen presenting cells (APC) that are important in regulatory immune responses. These APCs can process antigens and activate specific subsets of dendritic cells and Tregs which can travel to other sites in the body and program other cells of the immune system to suppress an immune response in the presence of antigen, in this case exogenously supplied FVIII. This phenomenon could be mediated by Fc interactions with FcRn and/or FcgR2b present on the APC of the gut mucosal immune system. Respiratory mucosa can be tolerized by inhalation of aerosols of rFVIIIFc which may operate based on the same mechanism as for the gut.

[00552] The ascertain whether immune tolerance can be achieve via mucosal route, XYNTHA* or ADVATE* will be administered orally or via aerosol to hemA mice. Following the induction of inhibitory antibodies, animals will be switched to rFVIIIFc. This will be the tolerization phase. Levels of anti-FVIII antibodies will be determined from blood samples drawn as indicated. Following the tolerization phase, animals will be injected again with XYNTHA* or ADVATE and blood samples drawn will be tested for anti-FVIII antibodies. In the event of successful tolerization, the second challenge with XYNTHA* or ADVATE should not produce any antibodies to FVIII which may indicate the capability of rFVIIIFc to induce immunotolerance to FVIII after mucosal administration.

Example 21 Immunotolerance to Other Clotting Factors 100553] To ascertain whether immunotolerance can be conferred by Fc to clotting factor payloads other than FVIII, chimeric polypeptides comprising the FVIIa clotting factor fused to Fc (rFVIIaFc), or the FIX clotting factor fused to Fe (rFIXF) are generated. Cloning, expression, and purification of rFVIIaFc and rFIXFc are performed according to methods known in the art. Biochemical, biophysical and pharmacokinetic characterization of rFVIIaFc and rFIXFc are conducted as disclosed in the examples above and/or using methods known in the art. The evaluation of antibody responses to rFVIlaFc and rFIXFc compared to commercial clotting factors and immunotolerance studies are performed as disclosed in the examples above. Chimeric polypeptides comprising a clotting factor payload and an Fe moiety such as rFIXFc or FVIIaFc can effectively induce immunotolerance to the unmodified clotting factor (i.e., FIX or FVII without an Fc portion).

Example 22 Transplacental Transfer of Immune Tolerance to FVIII using rFVIIIFc

[005541 The objective of this study was to determine whether the administration of rFVIIIFc to pregnant mice could transfer the molecule to the fetus via the placenta (due to the interaction of the Fe portion with the FcRn receptor on placental cells), and whether the exposure of the fetal immune system to the rFVIIIFe at an early stage could lead to tolerance by programming the thymus to recognize FVIII as a self antigen and not to develop an immune reaction towards it.

[005551 Pregnant mice were infused with either one dose (6U) of rFVIIIFe or XYNTHA* intravenously by retro-orbital injection on day 16 of gestation or two doses (6U each) by tail vein injection on days 15 and 17 of gestation. The immunogenicity of XYNTHA* was evaluated in the pups born out of the immunized mothers, after pups had reached maturity (ages 6-9 weeks old). Day 16 of pregnancy was chosen because it coincided with the development of the autoimmune regulatory molecule, AIRE, which plays a crucial role in removing self-reactive T-cells from the thymus. The results showed that in the first experiment, that tested dosing of pregnant mice with a single infusion of rFVIIIFc or XYNTHA* at day 16 of gestation, pups born out of rFVIIIFc treated pregnant mice had statistically significantly lower Bethesda titers to XYNTHA, compared to pups born out of XYNTHA* treated pregnant mice (FIG. 62). In the second experiment, that tested dosing of pregnant mice on d 15 and d17 of gestation, pups born out of rFVIIIFc treated pregnant mice had statistically significantly lower Bethesda titers to XYNTHA, compared to pups born out of control mothers and a trend for lower titers compared to pups born from mothers treated with XYNTHA* (FIG. 63)

[005561 The transplacental transfer of protein (rFVIIIFc) from maternal to the fetal circulation will be evaluated by detecting FVIII activity in pups born out of immunized mice. The actions at the T-cell level will be evaluated by T-cell profiling and Treg transfer studies from rFVIIIFc tolerized pups.

Example 23 rFVIIIFc Regulates Tolerogenic Markers in Antigen Presenting Cells

Materials and Methods:

[00557] Mice: Hemophilia A (HemA) mice (C57BL/6) bearing a FVIII exon 16 knockout on a 129 x B6 background (Bi, L., Lawler, A.M., Antonarakis, S.E., High, K.A., Gearhart, J.D., and Kazazian, H.H., Jr. 1995. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 10:119-121) were obtained from Dr. H. Kazazian (University of Pennsylvania, Philadelphia, PA, USA) and maintained as breeding colonies at either Biogen Idec Hemophilia or Charles River Laboratory or Jackson Laboratories. All animal procedures used were approved by the Institutional Animal Care and Use Committee (IACUC) and performed based on guidelines from the Guide to the Care and Use of Laboratory Animals.

[00558] Antibodies and Reagents: Antibodies used for staining and Flow Cytometry were obtained from BD Biosciences (Franklin Lakes, NJ, USA) or eBioscience (San Diego, CA, USA). Antibodies used were directed against murine surface markers such as CD4 and PD-I (T-helper cells), CD1ic, CD80, and PD-Li (dendritic cells), and CD25 (Treg cells), intracellular cytokines (IL-2, IL-4, TNF-a), and transcription factors (Foxp3). Reagents for intracellular staining for cytokines and transcription factors were purchased from BD Biosciences. Recombinant B-domain deleted FVIiIFc (rFVIIIFe) and recombinant B-domain deleted FVIII (rFVIII) were synthesized as in Peters et al. (Peters, R.T., Toby, G., Lu, Q., Liu, T., Kulman, J.D., Low, S.C., Bitonti, A.J., and Pierce, G.F. 2012. Biochemical and functional characterization of a recombinant monomeric Factor VIII-Fc fusion protein. J Thromb Haemost. DOI: 10.1111/jth.12076).

[00559] Other Factor VIII drug substances namely rBDD FVIII XYNTHA* (Wyeth Pharmaceuticals, Philadelphia, PA, USA), and full-length FVIII ADVATE* (Baxter Healthcare Corporation, Westlake Village, CA, USA) were purchased and reconstituted according to manufacturers' guidelines.

[00560] Immunization/Tolerance Induction in Mice: Three treatment groups consisting of 8 - 10 week old male HemA mice received intravenous doses of 50, 100, or 250 lU/kg, which were administered on days 0, 7, 14, 21, 35, and 53. Each treatment and dose level was administered to 10 mice. Blood samples were collected by retro-orbital bleeding on days 0 (pre-bleed), 14, 21, 28, and 42, and used for separating plasma and determining anti-FVIII antibody levels using ELISA. Animals were injected once more on day 53, euthanised on day 56 by CO 2 inhalation and spleens were dissected in sterile PBS. Splenocytes were separated using the mouse spleen dissociation kit and a gentle MACS dissociator (Miltenyi Biotec,

Cologne, Germany). Single cell suspensions of splenocytes were either fixed in 3% formalin (Boston BioProducts) for FACS staining or stored in dissociation buffer for RNA isolation (Roche Applied Science, Indianapolis, IN). For immune tolerance testing studies, mice were initially injected with 50 IU/kg on days 0, 7, 14, 21, and 35. After confirming the absence of antibodies to FVIII on day 42, these mice were administerd with 250 IU/kg of rFVIIIFc once weekly; i.e., days 49, 56, 63, and 70 (days 0, 7, 14, and 21 of rechallenge). Rechallenged animals were tested for anti-FVIII antibody levels on bleeds collected on days 14, 21, and 28.

[005611 Anti-FVIII antibody ELISA: The protocol followed was designed in house at Biogen Idec Hemophilia. Prior to the assay, all plasma samples were warmed to 56C in a water bath to inactivate residual coagulation factors introduced by the treatments and anti-coagulation enzymes that could break down the coated standards on the plate. On day 1, a 96 well, high binding microtiter plate (Thermo Immulon 2HB) was coated with 1 pg/ml (1001/well) B domain deleted FVIII in 0.05M sodium carbonate, pH 9.6 and incubated overnight (12 to 18 hours) at 4 °C. On the following day the supernatant was removed and the plate washed 4 times with PBST (phosphate buffered saline containing 0.05 % Tween 20). The plate was then blocked with 200 l per well of PBST containing 10% heat inactivated horse serum, pH 7.4 for 60 minutes at room temperature. The standard used for mouse IgG was a polyclonal pool of anti-FVIII monoclonal antibodies prepared by mixing equal amount of GMA8002 (Al), GMA8008 (C2), GMA8011(C1), GMA8015(A2), GMA8016 (A2), GMA8005 (AI/A3). All monoclonal antibodies were from Green Mountain Antibodies, Inc, Burlington, VT with the FVIII domain epitopes are shown in parenthesis. Mouse plasma test samples were diluted in blocking buffer and contained the same concentration of heated plasma as the standards. The blocking buffer was then removed and the diluted standards and samples were added at 100 pl/well in duplicates. The plate was incubated for 2 hours at 37 °C with shaking on an orbital shaker. After washing 4 times with PBST 100pl/well of goat anti-mouse IgG HRP, diluted 1:20000 in blocking buffer was added and incubated for 60 minutes at 37 °C with shaking on an orbital shaker. The plate was washed again 4 times with PBST, and then 100 pl/well of TMB was added and incubated at RT for 5 to10 minutes. Results were read at OD 650 with a plate reader

[00562] Bethesda Assay for Determining Neutralizing Antibody Titers: This assay determined the titer of neutralizing anti-FVIII antibodies in a given plasma sample. Briefly, the assay was performed by mixing plasma samples with known concentrations of recombinant FVII and incubation at 2 hours at 37°C. Residual FVIII activity in the mixture was then tested using a Coatest FVIII SP kit, in the presence of Factor IXa, Factor X, phospholipids and CaCl2. The activity of FVIII was calculated using a standard curve for FVIII activity plotted using rFVIII in the absence of any inhibitors.

[005631 FACS Staining: T-cell and dendritic cell response profiling was conducted on isolated mouse splenocytes. Splenic lymphocytes and dendritic cells were stained for surface and intracellular targets. For surface staining, lx106 total splenocytes were incubated with antibodies at appropriate concentrations. For intracellular staining, cells were permeabilised with BD Fix-Perm solution (BD Biosciences) followed by incubation with antibodies to cytokines in the same buffer. Foxp3 staining was carried out using antibody in Foxp3 staining buffer (BD Biosciences). Fluorescence intensity was recorded using a BD FACS Canto II and analysis performed using FLOWJO software. Lymphocytes were costained with CD4 and intracellular markers IL-2, TNF-a, and IL-4. Regulatory T cells (Treg) were stained for surface markers CD4 and CD25 followed by intracellular Foxp3. Splenocytes were stained for CDlic and PD-L1 (dendritic cells) or CD4 and PD-1 (CD4+ T-cells) to identify cells involved in the PD-Li- PD-1 pathway.

[00564J Real Time PCR and Real time PCR based Array Analysis: Total RNA was isolated using an RNA isolation kit (Roche Applied Science, Indianapolis, IN) and reverse transcribed to cDNA (Qiagen, Hilden, Germany). Real time PCR primers for the tested genes were designed using the online algorithm available on the IDT technologies website (www.idtdna.com) and were purchased from IDT technologies (Coralville, IA). SYBR green-based real-time PCR was carried out using Quantitect system (Qiagen, Hilden, Germany) in an ABI 7900 Fast Block real-time PCR machine (Applied Biosystems, Foster City, CA). For real time PCR based arrays, 500 ng of total cDNA was mixed with SYBR green based qPCR master mix, and aliquoted onto 96-well plate array (PAMM047Z, T-cell Anergy and Immune Tolerance PCR Array; SA Biosciences, Frederick, MD, USA). Reactions were carried out with an ABI 7900 Fast Block real-time PCR machine (Applied Biosystems, Foster City, CA). Results were analysed using the 7500 software version 2.0.5 and the relative levels of gene transcripts were measured with endogenous housekeeping genes as control using the 2-ACt relative quantification method (Livak, K.J., and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.). The housekeeping genes used for normalization were GAPDH, HPRT, Hsp90ab, beta-actin, and GusB. For each sample average Ct values for the housekeeping genes were taken together and used to calculate the ACt. mRNAs that displayed threshold cycles (Ct) >35 were excluded from the analysis.

[005651 T-cell Proliferation and Determination of Interferon-y (IFN-y) levels: HemA mice (8-10 weeks old) were injected with FVII drug substances once a week for 2 weeks. Seventy two hours post the second injection mice wereeuthanized and peritoneal macrophages were collected by lavage using sterile PBS. Splenic T-cells were isolated using magnetic bead based murine CD4+ T-cell isolation kit (Miltenyi Biotec, Germany). T-cells were labeled with 10 M CFSE (Invitrogen, Carlsbad, CA) for 15 minutes in warm PBS and plated in 96 well ultra-low adhesion plates (Corning) along with peritoneal macrophages at a density of 1xi06 cells per ml for each. Cells were then incubated with 0.1, 1 and 10 nM of rFVIII or vehicle or CD3/CD28 microbeads (positive control; Miltenyi Biotec) in X-VIVO 15 medium (Lonza) containing co-stimulatory antibodies namely anti-CD28 and anti-CD49d (BD Biosciences), for 96 hours at 37 °C. At the end of the incubation, IFNy levels in the culture supernatant were measured using an ELISA kit from Meso Scale Devices (MSD). T-cell proliferation was determined by measuring CFSE fluorescence intensity (MFI) in T-cells gated based on forward and side scatters, using FACS (BD FACS CANTO II).

[00566] Statistical Analysis: Statistical analysis of results were carried out either using unpaired student's T-test or Mann-Whitney's T-test. P-values <0.05 were considered to be significant.

[00567] Results: Dendritic cells are professional antigen presenting cells and harbor key molecules and enzymes that are pivotal in skewing an immune response towards tolerance

. Splenocytes from hemA mice injected with FVIII drug substances or vehicle were stained for CD11c and MHC Class II molecules to gate the dendritic cells. Splenic composition of dendritic cells expressing markers such as CD80, a surface marker upregulated in mature dendritic cells indicating more antigen presentation and CD274 (PD-L), the ligand for the PD-1 receptor, were identified by co-staining with specific antibodies to these molecules. As shown in FIG. 63A, splenocytes from 100 IU/kg rFVIIIFc or BDD-FVIII injected HemA mice showed a significant decrease in the percentage of dendritic cells expressing CD80, suggesting an abundance of immature dendritic cells.

[00568] Although mice receiving fl-FVIII showed a decrease in the percentage of CD80+ dendritic cells compared to vehicle, it did not attain statistical significance (FIG. 64A). The trend was similar for mice receiving the 50 IU/kg dose of rFVIIIFc. At 250 IU/kg, all the three treatments showed no alterations in the percentage of CD80+ dendritic cells among the splenocytes.

[00569] Results from FACS analysis for splenic dendritic cells expressing CD274 (PD Li), revealed that rFVIIIFc at 100 IU/kg enhanced the percentage of these cells compared to vehicle and other FVIII treatments (FIG. 64B). This molecule was also regulated at the mRNA level by rFVIIIFc as illustrated by real time PCR analysis (FIG. 64C). This suggests the rFVIIIFc regulates the PD-L:PD-1 pathway, one of the key immunosuppressive pathways, thereby reducing the immunogenicity to FVIII. In addition to CD274, rFVIIIFc also upregulated mRNA levels of indoleamine 2,3-dioxygenase (IDO), a key enzyme that regulates T-cell proliferation and activation by affecting tryptophan metabolism (FIG. 64D).

Example 24 rFVIIIFc at Low Doses Enhances the Expression of Markers of Immune Tolerance and Anergy in Splenocytes

[00570] Experimental procedures were conducted according to the Materials and Methods described in Example 23. 1005711 In order to identify markers of immune tolerance induced by rFVIIIFc, real time PCR based arrays focused on genes specific for immune tolerance and anergy (SA Biosciences) was employed. mRNA isolated from splenocytes obtained from mice injected with 50 and 250 IU/kg of rFVIIIFc and vehicle was used to identify novel response elements activated by this drug substance.

[00572] Candidates were identified based on their expression level (2-fold above or below vehicle), and the p-value based on student's T-test (<0.05). Relative expression of each gene was determined using the 2-Act method (Livak, K.J., and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408) after normalizing to the average Ct values of a set of 5 housekeeping genes (see Example 23, Materials and Methods).

[00573] Based on these criteria, array analysis performed revealed candidates that were preferentially regulated by rFVIIIFc at 50 IU/kg at the splenocyte level, in comparison to vehicle and 250 IU/kg injected mice (FIG. 65 and FIG. 66). These included statistically significant upregulation of tolerance specific genes such as Foxp3, CTLA-4, and CD25; anergy associated genes such as Egr2, Dgka, and CBL-B; genes belonging to the Tumor

Necrosis Factor Superfamily (TNFRSF); prostaglandin synthase 2 (PTGS2) and prostaglandin E2 receptor (PTGER2) (see FIG. 66). Conversely some of the downregulated genes identified using this array are known pro-inflammatory molecules such as CCL3 and STAT3 (FIG. 66).

[005741 These results indicated the presence of tolerogenic pathways and a tolerogenic microenvironment within the splenocytes of animals receiving 50 IU/kg of rFVIIIFc. The gene expression profiles determined using the array will be validated using real time PCR with individual primer pairs for the identified candidates.

Example 25 CD4+ Splenocytes from 250 IU/kg Group of Mice Proliferate and Produce High Levels of IFN-y

[005751 Experimental procedures were conducted according to the Materials and Methods described in Example 23.

[005761 T-cell proliferation studies were employed to investigate factor VIII specific T-cell responses ex vivo. CD4+ T-cells isolated from mice receiving two weekly injections of either 50 or 250 IU/kg of rFVIIIFc, were labeled with CFSE and reconstituted with peritoneal macrophages (antigen presenting cells; APC) and incubated in the presence of B-domain deleted rFVIII at three concentrations, 0.1, 1, and 10 nM.

[005771 FACS analysis for proliferation based on CFSE dilution signals revealed that, T-cells from the group treated with 250 IU/kg of rFVIIIFc showed a dose dependent increase in proliferation, which was statistically significant at 10 nM (FIG. 67). In parallel, T-cells from mice treated with 50 IU/kg of rFVIIIFc did not show a significant increase in proliferation with escalating concentrations of rFVIII ex vivo compared to vehicle (FIG. 67).

[00578] IFN levels measured from the culture supernatants of these incubations revealed a similar profile matching the T-cell proliferation pattern, i.e., a dose dependent increase in secretion from T-cells derived from mice treated with 250 IU/kg of rFVIIIFc (FIG. 68A) whereas no changes in levels from T-cells from mice treated with 50 IU/kg of rFVIIIFc (FIG. 68B). 1005791 In order to dissect the mechanism(s) of action of rFVIIIFc in suppressing the immune response to FVIII, we employed two mutant constructs - one which does not bind to the Fcy receptor (termed rFVIIIFc-N297A) and the other one which lacks binding the to the FcRn receptor (termed rFVIIIFe-IHH). These constructs were used to identify the interactions of the rFVIIIFc with one of these receptors in bringing about the immunosuppressive action. To this end, we tested the IFNy secretion profile of T-cells derived from mice that received two weekly injections of rFVIIIFc-N297A at 250 IU/kg doses (FIG. 68C) and 50 IU/kg doses (FIG. 68D).

[005801 Side by side comparisons of IFNy secretion from T-cells derived from 250 IU/kg of rFVIIIFc and rFVIIIFc-N297A revealed that the level of the cytokine was highly reduced in the T-cells from animals receiving the mutant, albeit significantly higher levels compared to vehicle. However, the levels of IFNy from the 50 IU/kg group of the mutant protein did not show a significant difference from that of 50 IU/kg of rFVIIIFc. This suggests that the higher antibody production and T-cell proliferation observed at higher doses of rFVIIIFc might be a resultant of the interaction of the Fc with the Fey receptors which is abolished by the Fey non binding mutant.

[00581J The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

TABLE 1: Polynucleotide Sequences

A. B-Domain Deleted FVILIFc

(i) B-Domain Deleted FVIIIFc Chain DNA Sequence (FVIII signal peptide underlined, Fe region in bold) (SEQ ID NO:1, which encodes SEQ ID NO:2) 661 A TGCAAATAGA GCTCTCCACC TGCTTCTTTC 721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT CCAGTGGAAC 781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC 841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG 901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC 961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG 1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTCTATCCTA CTGCGAACCT TCTGAGGGAG 1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG

1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATCG TCCAATGGCC TCTGACCCAC 1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261 TCATTGGAGC CCTACTAGTA TGTACAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA 1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTOC TCGGGCCTGG CCTAAAATGC 1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501 CACTCTATTG CCATCTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA ACTAGACAGC TGTCCAGAGG 1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG 1801 ATTCTGAAAT GGATGTGOTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA ACTCCGATTT ATGGCATACA 2041 CAGATGAAAC CTTTAAGACT CGTCAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCCCT 2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GCCCCTCTCC 2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA CATAATGTCA GACAAGAGGA 2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTC GTACCTCACA GAGAATATAC 2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTOGCA 3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGOCTCC TTTACTCAGC 3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961 GAAAAAAACA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG 4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGCCACCAAT GATTATTCAC GGCATCAAGA 4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441 TCTTTGGCAA TGTGGATTCA TCTCGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT COCATGGAGT 4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCACCAGTC 4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATCGCAA AGTAAAGGTT TTTCAGGGAA 4921 ATCAAGACTC CTTCACACCT GTGOTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041 GCGAGOCACA GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC

5101 TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 5161 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA 5221 AGTTCAACTG GTACGTGGAC GGCCTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG 5281 AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC 5341 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 5401 AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT 5461 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC 5521 CCAGCGACAT CGCCGTGGAG TCGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA 5581 CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA 5641 AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA 5701 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A

(ii) Fe DNA sequence (mouse IgK signal peptide underlined) (SEQ ID NO:3, which encodes SEQ ID NO:4)

7981 ATGGA GACAGACACA 8041 CTCCTGCTAT GGGTACTGCT CCTCTGGGTT CCAGCTTCCA CTGGTGACAA AACTCACACA 8101 TGCCCACCGT GCCCAGCACC TGAACTCCTG GGAGCACCGT CAGTCTTCCT CTTCCCCCCA 8161 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 8221 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT 8281 AATGCCAAGA CAAACCCCCC GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC 8341 CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 8401 AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 8461 CCACAGGTGT ACACCCTGCC CCCATCCCGC GATGAGCTCA CCAAGAACCA GGTCAGCCTG 8521 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG 8581 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC 8641 CTCTACAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC 8701 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 8761 GGTAAA

B, Full Length FVIIIFc

(i) Full Length FVIIIFc DNA Sequence (FVIIl signal peptide underlined, Fc region in bold) (SEQ ID NO:5, which encodes SEQ ID NO:6)

661 ATG CAAATAGAGC TCTCCACCTG 721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC 781 AGTGGAACTG TCATCGCACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG 841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC 901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT 961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA 1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGCT GTATCCTACT GGAAAGCTTC 1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT 1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC 1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA 1261 TTCAGGCCTC ATTGGACCOC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC 1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA 1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC 1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA 1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT 1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC 1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT 1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG 1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATCAACAA GCGGAAGACT ATGATGATGA 1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT 1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA 1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG 1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT 2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT 2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTCTTG ATTATATTTA AGAATCAAGC 2161 AACCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG 2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT

2281 CAAATATAAA TGGACACTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAOCTTCAG GACTCATTGG 2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA 2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA 2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT 2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC 2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA 2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC 3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC 3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACACTAATAA 3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT 3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGCGGACAA CTGCAGCAAC 3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT 3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541 TACTGAGTCT GGTCGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA 3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG 3661 TGGTAGOTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC 3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT CAGAATAGTC CATCAGTCTG 3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC 3961 TTCATCAAAA AACATGGAAA TGCTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC 4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT 4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA 4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT 4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT 4441 ACTGACCACT ACGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA 4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA 4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA 4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC 4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861 CTTATCAGAT TGCCTTACCA CGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA 5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC 5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC 5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341 AGAGGGAGCG ATTAAGTGGA ATCAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT 5401 AGCAACAGAA ACCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA 5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA 5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA CAAGTCACCT GGGCAAAGCA 5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC 5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG 5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA 5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG 6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA 6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT 6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC 6181 TAATGAAACC AAAACTTACT TTTGGAAAGT CCAACATCAT ATGGCACCCA CTAAAGATGA

6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC 6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG 6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG 6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA 6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT 6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG 6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA 6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT 6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG 6781 CATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC 6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGCCCCAAA 6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC 6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC CGCATCAAGA CCCAGGGTGC 7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG 7081 GAAGAACTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA 7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT 7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG 7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA 7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGCTCTCCTT CAAAAGCTCG 7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG 7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA 7501 ATCTCTGCTT ACCAOCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA 7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC 7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA 7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT OCCAGOCACA 7741 GGACCTCTAC GACAAAACTC ACACATCCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG 7801 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 7861 TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 7921 GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA 7981 CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 8041 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 8101 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 8161 GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 8221 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 8281 GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG B341 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 8401 GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A

(ii) Fe (same sequence as A (ii) (SEQ ID NO:3))]

TABLE 2: Polypeptide Sequences

A. B-Domain Deleted FVIII-Fe Monomer Hybrid (BDD FVIIIFc monomer dimer): created by coexpressing BDD FVIIIFc and Fe chains.

Construct = HC-LC-Fc fusion. An Fe expression cassette is cotransfected with BDDFVIII-Fc to generate the BDD FVIIIFc monomer-. For the BDD FVIIIFc chain, the Fe sequence is shown in bold; HC sequence is shown in double underline; remaining B domain sequence is shown in italics. Signal peptides are underlined.

i) B domain deleted FVIII-Fc chain (19 amino acid signal sequence underlined) (SEQ ID NO:2)

MQIELSTCFFLCLLRFCFS ATRRYYLGAVELSWDYMOSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVI TLKNMASHPVSLHAVGVSYWKASEGAEYDDOTSQREKEDDKVFPGCSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL IGAL.LVCREGSLAKEKT LHKFILLFAVFDEGKSWH SETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVI GMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEE PLRMKNNEEAE DYDDDLTDSEMDVVRFDDDNSPSFIOIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDETFKTREAIQHESCILGPLLYGEVGDTLLIIFKN ASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVT VEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQ LEDPEFQASNIMHSINGYVFDSLOLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLW ILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHOREIT RTTLQSDQEEIDYDDTI SVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLW DYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIM DTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMST LFLVYSNKCQTPLGMASCHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI SQFIIMYSLDGKKWQTYRGNSTCTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKA TSDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQ WTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVHEALHNHYTQKSLSLSPGK

ii) FE chain (20 amino acid heterologous signal peptide from mouse Ig chain underlined) (SEQ ID NO:4)

METDTLLLWVLLLWVPGSTG DKTHTCPPCPAPELLGCPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

B. Full length FVIIIFc monomer hybrid (Full length FVIIIFc monomer dimer): created by coexpressing FVIIIFc and Fc chains.

Construct = HC-B-LC-F fusion. An Fe expression cassette is cotransfected with full length FVIII-FE to generate the full length FVIFc monomer. For the FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; B domain sequence is shown in italics. Signal peptides are underlined.

i) Full length FVIIlFc chain (FVIII signal peptide underlined (SEQ ID NO:6)

MQTELSTCFFLCLLRFCFS ATRRYYLGAVELSWDYMOSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAlVYDTVVI TLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSOREKEDDKVFPGGSHTYVWQOVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLICCHRKSVYWHVI GMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTA TLLMDLGQFLLFCHTSSHQHDGMFAYVKVDSCPEEPQLRMKNNEEAE DYDDDLTDSEMDVVRFDDDNSPSFIOIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSOYLNNGPQRIGRKYKKVRFMA

YTDETFKTREAIQHESGILGPLLYGEVGDTLL IIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVT VEDGPTKSDPRCLTRYYSSFVNMERDLASCLIGPLLICYKESVDQRGNCIMSDKRNVILFSVFDENRSWYLTENIORFLPNPAGVQ LEDPEFOASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLW ILGCHNSDFRNRGMTALLKVSSCDKNTGDYYE DSYE DISAYLLSKNNATE PRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFA HRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYET FSDDPSPGAIDSNNJSLSEMTHFRPQLHHSGDMVFTPESGLQLRLN EKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSK LLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNI LESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSL NSGQGPSPKQLVSLGPEKSVEGQANFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETL IQENVVLPQIH TVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNR TKKHTAHFSKKGEEENLEGLGNQTKQIVEK YACTTRISFNTSQQNFVTQRSKRALKQFRL FLEE TELEKRIIVDDTSTQWSKNMKHLTST LTQIDYNEKEKGAI TQSPLSDCL TR SHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSL GTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSWGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLR VATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTER LCSQNFPVLKRHOREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNF VKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNL YPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWST KEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR LHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQTTASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQK TMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME VLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTMISRTPEVTCVVVDVSHEDPEVKFNYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ii) Fc chain (20 amino acid heterologous signal peptide from mouse IgK chain underlined) (SEQ ID NO:4)

METDTLLLWVLLLWVPGSTG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[005821 In a first embodiment there is provided a method of reducing an inhibitory Factor VIII (FVIII) immune response in a subject in need thereof, comprising administering to the subject a composition comprising a chimeric polypeptide comprising a FVIII portion and a first Fc portion, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1475 of SEQ ID NO: 2.

[00583] In a second embodiment there is provided a method of preventing or inhibiting development of an inhibitor to FVIII in a subject in need thereof, the method comprising administering to the subject a composition comprising a chimeric polypeptide comprising a FVIII portion and a first Fc portion, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1457 of SEQ ID NO: 2.

[00584] In a third embodiment there is provided a method for inducing immune tolerance to FVIII in a subject, wherein the subject is a fetus, wherein the chimeric polypeptide is suitable for administration to the mother of the fetus, and wherein the polypeptide comprises a FVIII portion and an Fc portion, wherein the FVIII portion consists essentially of or consists of the A2 domain of FVIII, the C2 domain of FVIII, or the A2 domain and the C2 domain of FVIII.

[00585] In a fourth embodiment there is provided a kit when used for reducing an inhibitory immune response to FVIII, said kit comprising: (a) a pharmaceutical composition comprising a chimeric polypeptide which comprises a FVIII portion and a first Fc portion and a pharmaceutically acceptable carrier, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1457 of SEQ ID NO: 2 and wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2, and (b) instructions to administer the composition to a subject in need of reducing an inhibitory immune response to FVIII.

[00586] In a fifth embodiment there is provided the use of a chimeric polypeptide comprising a FVIII portion and a first Fc portion, in the manufacture of a medicament for reducing an inhibitory Factor VIII (FVIII) immune response in a subject, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1475 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

[005871 In a sixth embodiment there is provided the use of a chimeric polypeptide comprising a FVIII portion and a first Fc portion, in the manufacture of a medicament for preventing or inhibiting development of an inhibitor to FVIII in a subject, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

[00588] In a seventh embodiment there is provided the use of a chimeric polypeptide in the manufacture of a medicament for inducing immune tolerance to FVIII in a subject, wherein the subject is a fetus, wherein the medicament is suitable for administration to the mother of the fetus, and wherein the polypeptide comprises a FVIII portion and an Fc portion, wherein the FVIII portion consists essentially of or consists of the A2 domain of FVIII, the C2 domain of FVIII, or the A2 domain and the C2 domain of FVIII.

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

SEQUENCE LISTING <110> Biogen Idec MA Inc. Jiang, Haiyan Liu, Tongyao Krishnamoorthy, Sriram Josephson, Neil <120> METHODS OF REDUCING IMMUNOGENICITY AGAINST FACTOR VIII IN INDIVIDUALS UNDERGOING FACTOR VIII THERAPY <130> 2159.371PC02/EKS/C-K/JCA 2018267631

<140> To be assigned <141> 2013-01-11

<150> US 61/668,961 <151> 2012-07-06 <150> US 61/586,103 <151> 2012-01-12 <160> 6 <170> PatentIn version 3.5

<210> 1 <211> 5052 <212> DNA <213> Homo sapiens

<400> 1 atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60 accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180 acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420 gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480 aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660 tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720 gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780 ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840 accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960 Page 1

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080 gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140 gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260 cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320 2018267631

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440 ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620 actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680 gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740 agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920 tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280 ttctctcaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340

cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400 gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460 cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520 catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580 caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640 ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700 agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760 cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820

Page 2

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880 gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940 ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000

tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060 gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120

tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180 caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240 2018267631

attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300 tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360 tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420

gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480 cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540 tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600

ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660 ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720

cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780

aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840 tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900

atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960 tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020

agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080

aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140

tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200 cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260 tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320

cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta cgacaaaact 4380 cacacatgcc caccgtgccc agctccagaa ctcctgggcg gaccgtcagt cttcctcttc 4440 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 4500 gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 4560 gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 4620 agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 4680 tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 4740 Page 3

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 4800

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 4860 aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgttggactc cgacggctcc 4920 ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 4980 tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 5040 tctccgggta aa 5052 2018267631

<210> 2 <211> 1684 <212> PRT <213> Homo sapiens <400> 2

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile 65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 Page 4

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 2018267631

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Page 5

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 2018267631

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670

Page 6

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 2018267631

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780

Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800

Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815

Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830

Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845

Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880

Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895

Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910

Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925

Page 7

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940

Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960

Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 2018267631

Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990

Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005

Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1010 1015 1020

Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1025 1030 1035

Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1040 1045 1050

Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1055 1060 1065

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1070 1075 1080

Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095

Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110

Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120 1125

Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140

Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1145 1150 1155

Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1160 1165 1170 Page 8

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1175 1180 1185

Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200

Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215 2018267631

Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230

Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245

Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1250 1255 1260

Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1265 1270 1275

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1280 1285 1290

Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1295 1300 1305

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320

Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335

Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350

Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360 1365

Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380

Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1385 1390 1395

Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Page 9

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

1400 1405 1410

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1415 1420 1425

Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440

Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1445 1450 1455 2018267631

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1460 1465 1470

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1475 1480 1485

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1490 1495 1500

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1505 1510 1515

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1520 1525 1530

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1535 1540 1545

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 1550 1555 1560

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1565 1570 1575

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1580 1585 1590

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 1595 1600 1605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1610 1615 1620

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1625 1630 1635

Page 10

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1640 1645 1650

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1655 1660 1665

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1670 1675 1680

Lys 2018267631

<210> 3 <211> 741 <212> DNA <213> Homo sapiens

<400> 3 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggagg accgtcagtc 120

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 180 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 240

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 300

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 360 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 420

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgcgatga gctgaccaag 480 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 540

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gttggactcc 600

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 660 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 720

ctctccctgt ctccgggtaa a 741

<210> 4 <211> 247 <212> PRT <213> Homo sapiens <400> 4 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15

Gly Ser Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30

Page 11

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 65 70 75 80 2018267631

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 145 150 155 160

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240

Leu Ser Leu Ser Pro Gly Lys 245

<210> 5 <211> 7734 <212> DNA <213> Homo sapiens <400> 5 Page 12

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60 accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120 ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240 gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360 ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420 2018267631

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480 aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540 gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660 tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720 gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840 accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020 gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140 gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380 attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440 ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560 ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620 actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680 gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740 agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860 cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920 Page 13

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040 atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160 atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220 agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280 2018267631

ttctcccaga attcaagaca ccctagcact aggcaaaagc aatttaatgc caccacaatt 2340

ccagaaaatg acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa 2400 atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc tactccacat 2460 gggctatcct tatctgatct ccaagaagcc aaatatgaga ctttttctga tgatccatca 2520

cctggagcaa tagacagtaa taacagcctg tctgaaatga cacacttcag gccacagctc 2580 catcacagtg gggacatggt atttacccct gagtcaggcc tccaattaag attaaatgag 2640 aaactgggga caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca 2700 tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac tgataataca 2760

agttccttag gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta 2820

tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt gagtgaagaa 2880 aataatgatt caaagttgtt agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940

aaaaatgtat cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000

gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt aaagacaaac 3060 aaaacttcca ataattcagc aactaataga aagactcaca ttgatggccc atcattatta 3120

attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180

gtgacacctt tgattcatga cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240 aatcatatgt caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300

gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360 ttgccagaat cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg 3420 caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480 aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540 ggactcaaag agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat 3600 ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660 gaaacattaa tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag 3720 aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg ttcatatgac 3780

Page 14

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

ggggcatatg ctccagtact tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840 aagaaacaca cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900 aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc tcctaataca 3960

agccagcaga attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca 4020 ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac ccagtggtcc 4080

aaaaacatga aacatttgac cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140 aaaggggcca ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct 4200 2018267631

caagcaaata gatctccatt acccattgca aaggtatcat catttccatc tattagacct 4260 atatatctga ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat 4320 agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380

aacctttctt tagccattct aaccttggag atgactggtg atcaaagaga ggttggctcc 4440 ctggggacaa gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500 aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560

cagaaggacc tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620 gaagggagcc ttcttcaggg aacagaggga gcgattaagt ggaatgaagc aaacagacct 4680

ggaaaagttc cctttctgag agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740

ttggatcctc ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa 4800 tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860

aacgcttgtg aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920 atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980

gtcttgaaac gccatcaacg ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040

attgactatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 5100

gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160 gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 5220 gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 5280

tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340 tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 5400 ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 5460 cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 5520 catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580 gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 5640 acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 5700 Page 15

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760

ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 5820 aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 5880 tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 5940 gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 6000 gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060 2018267631

attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 6120

cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 6180 caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 6240 tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 6300

cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360 atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 6420 accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 6480 cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 6540

cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 6600

agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 6660 acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720

caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780

ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840 atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900

gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 6960

ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 7020 gaggttctgg gctgcgaggc acaggacctc tacgacaaaa ctcacacatg cccaccgtgc 7080

ccagctccag aactcctggg cggaccgtca gtcttcctct tccccccaaa acccaaggac 7140 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 7200 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 7260 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 7320 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 7380 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 7440 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 7500 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 7560

Page 16

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

aactacaaga ccacgcctcc cgtgttggac tccgacggct ccttcttcct ctacagcaag 7620 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 7680 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaa 7734

<210> 6 <211> 2578 <212> PRT <213> Homo sapiens <400> 6 2018267631

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile 65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Page 17

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 2018267631

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445

Page 18

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 2018267631

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700

Page 19

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 2018267631

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 755 760 765

Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp 770 775 780

Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys 785 790 795 800

Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser 805 810 815

Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr 820 825 830

Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn 835 840 845

Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly 850 855 860

Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu 865 870 875 880

Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys 885 890 895

Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn 900 905 910

Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met 915 920 925

Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys 930 935 940

Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu 945 950 955 960 Page 20

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu 965 970 975

Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe 980 985 990

Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala 995 1000 1005 2018267631

Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser 1010 1015 1020

Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser 1025 1030 1035

Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu 1040 1045 1050

Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg 1055 1060 1065

Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met 1070 1075 1080

Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln 1085 1090 1095

Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met 1100 1105 1110

Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile 1115 1120 1125

Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro 1130 1135 1140

Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu 1145 1150 1155

Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys 1160 1165 1170

Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro 1175 1180 1185

Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Page 21

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

1190 1195 1200

Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu 1205 1210 1215

Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile 1220 1225 1230

His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu 1235 1240 1245 2018267631

Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr 1250 1255 1260

Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn 1265 1270 1275

Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu 1280 1285 1290

Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu 1295 1300 1305

Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln 1310 1315 1320

Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg 1325 1330 1335

Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp 1340 1345 1350

Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro 1355 1360 1365

Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala 1370 1375 1380

Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser 1385 1390 1395

Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser 1400 1405 1410

Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe 1415 1420 1425

Page 22

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1430 1435 1440

Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys 1445 1450 1455

Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly 1460 1465 1470

Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser 2018267631

1475 1480 1485

Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp 1490 1495 1500

Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His 1505 1510 1515

Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser 1520 1525 1530

Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr 1535 1540 1545

Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val 1550 1555 1560

Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser 1565 1570 1575

Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln 1580 1585 1590

Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys 1595 1600 1605

Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys 1610 1615 1620

Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys 1625 1630 1635

Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg 1640 1645 1650

Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu 1655 1660 1665

Page 23

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1670 1675 1680

Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1685 1690 1695

Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1700 1705 1710 2018267631

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1715 1720 1725

Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 1730 1735 1740

Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 1745 1750 1755

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1760 1765 1770

His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1775 1780 1785

Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1790 1795 1800

Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1805 1810 1815

Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr 1820 1825 1830

Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu 1835 1840 1845

Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 1850 1855 1860

Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His 1865 1870 1875

Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln 1880 1885 1890

Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1895 1900 1905 Page 24

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1910 1915 1920

Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1925 1930 1935

Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1940 1945 1950 2018267631

Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1955 1960 1965

Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1970 1975 1980

Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1985 1990 1995

Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 2000 2005 2010

Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 2015 2020 2025

Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 2030 2035 2040

Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala 2045 2050 2055

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His 2060 2065 2070

Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 2075 2080 2085

Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 2090 2095 2100

Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser 2105 2110 2115

Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 2120 2125 2130

Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Page 25

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

2135 2140 2145

Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2150 2155 2160

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 2165 2170 2175

Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 2180 2185 2190 2018267631

Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 2195 2200 2205

Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 2210 2215 2220

Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 2225 2230 2235

Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 2240 2245 2250

Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 2255 2260 2265

Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 2270 2275 2280

Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys 2285 2290 2295

Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 2300 2305 2310

Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His 2315 2320 2325

Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 2330 2335 2340

Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro 2345 2350 2355

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 2360 2365 2370

Page 26

2159_371PCTsequence_listing_ST25.txt 22 Nov 2018

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 2375 2380 2385

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 2390 2395 2400

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 2405 2410 2415

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 2018267631

2420 2425 2430

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 2435 2440 2445

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 2450 2455 2460

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 2465 2470 2475

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 2480 2485 2490

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 2495 2500 2505

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 2510 2515 2520

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 2525 2530 2535

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 2540 2545 2550

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 2555 2560 2565

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 2570 2575

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Claims (44)

1. A method of reducing an inhibitory Factor VIII (FVIII) immune response in a subject in need thereof, comprising administering to the subject a composition comprising a chimeric polypeptide comprising a FVIII portion and a first Fc portion, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1475 of SEQ ID NO: 2.

2. The method of claim 1, wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

3. The method of claim 1 or claim 2, wherein the FVIII portion comprises an amino acid sequence at least 99% identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence at least 99% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

4. The method of any one of claims 1 to 3, wherein the FVIII portion comprises an amino acid sequence identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence identical to amino acids 1458 to 1683 of SEQ ID NO: 2.

5. A method of preventing or inhibiting development of an inhibitor to FVIII in a subject in need thereof, the method comprising administering to the subject a composition comprising a chimeric polypeptide comprising a FVIII portion and a first Fc portion, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1457 of SEQ ID NO: 2.

6. The method of claim 5, wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

7. The method of claim 5 or claim 6, wherein the FVIII portion comprises an amino acid sequence at least 99% identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence at least 99% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

8. The method of any one of claims 5 to 7, wherein the FVIII portion comprises an amino acid sequence identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence identical to amino acids 1458 to 1683 of SEQ ID NO: 2.

9. The method of any one of claims 1 to 8, wherein the FVIII portion is covalently linked through its carboxy-terminus to the N-terminus of the first Fc portion.

10. The method of any one of claims 1 to 9, wherein the first Fc portion forms a disulfide bond with a second Fc portion, wherein the second Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

11. The method of any one of claims 1 to 10, wherein the first Fc portion forms a disulfide bond with a second Fc portion, wherein the second Fc portion comprises an amino acid sequence identical to amino acids 1458 to 1683 of SEQ ID NO: 2.

12. The method of any one of claims 1 to 11, wherein the subject is at risk of developing an inhibitory immune response against FVIII if administered an equivalent dose of FVIII without an Fc portion.

13. The method of any one of claims I to 12, wherein the subject has developed an inhibitory immune response against FVIII or has never been previously treated with FVIII.

14. The method of any one of claims I to 13, wherein the subject is a child or an adult.

15. The method of claim 14, wherein the child is less than one year old.

16. The method of any one of claims 1 to 15, wherein the FVIII portion comprises one or more half-life extending moieties other than the first Fc portion.

17. The method of any one of claims 10 to 16, wherein the inhibitory immune response comprises production of inhibitory antibodies to FVIII.

18. The method of claim 17, wherein the antibody concentration is at least 0.6 Bethesda Units (BU).

19. The method of claim 17, wherein the antibody concentration is at least 5.0 Bethesda Units (BU).

20. The method of any one of claims 12 to 19, wherein the inhibitory immune response comprises a cell-mediated immune response.

21. The method of claim 20, wherein the cell-mediated immune response comprises release of a cytokine selected from IL-12, IL-4, and TNF-a.

22. The method of any one of claims 12 to 21, wherein the inhibitory immune response comprises a clinical symptom selected from the group consisting of: increased bleeding tendency, high FVIII consumption, lack of response to FVIII therapy, decreased efficacy of FVIII therapy, and shortened half-life of FVIII.

23. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the number of antibodies to FVIII in the subject or the titer of antibodies to FVIII in the subject, or reduces the level of a cytokine in the subject compared to the level that would result from administration of polypeptide consisting of the FVIII portion to the subject.

24. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the titer of anti-clotting factor antibodies in the subject compared to the titer that would result from administration of a polypeptide consisting of the FVIII portion to the subject.

25. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the number of anti-clotting factor antibodies in the subject compared to the number that would result from administration of a polypeptide consisting of the FVIII portion to the subject.

26. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the number of antibodies to FVIII in the subject compared to the number in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

27. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the titer of antibodies to FVIII in the subject compared to the titer in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

28. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the level of a cytokine in the subject.

29. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the level of a cytokine in the subject compared to the level in the subject after a previous treatment with a polypeptide consisting of a FVIII polypeptide.

30. The method of any one of claims 1 to 22, wherein the chimeric polypeptide reduces the level of a cytokine in the subject compared to the level that would result from administration of a polypeptide consisting of the FVIII portion to the subject.

31. The method of any one of claims 1 to 30, wherein the subject is identified to have one or more characteristics selected from the group consisting of: (a) has a mutation or deletion in the gene encoding FVIII; (b) has a rearrangement in the gene encoding FVIII; (c) has a relative who has previously developed an inhibitory immune response against FVIII; (d) is receiving interferon therapy; (e) is receiving anti-viral therapy; (f) has a genetic mutation in a gene other than the gene encoding FVIII which is linked with an increased risk of developing an inhibitory immune response; and (g) any combination thereof.

32. The method of claim 31, wherein the genetic mutation in a gene other than the gene encoding FVIII comprises one or more mutations selected from the group consisting of:

(i) a genetic polymorphism associated with increased TNF-a; (ii) a genetic polymorphism associated with increased IL-10; (iii) a genetic polymorphism associated with decreased CTLA-4; (iv) a mutation in DR15 or DQB0602 MHC Class II molecules; and (v) any combination thereof.

33. The method of any one of claims 1 to 32, wherein the chimeric polypeptide is a monomer dimer hybrid comprising a first chain comprising the FVIII portion and the first Fc portion, and a second chain consisting essentially of or consisting of a second Fc portion, wherein the FVIII portion comprises an amino acid sequence identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence identical to amino acids 1458 to 1683 of SEQ ID NO: 2, and wherein the second Fc portion comprises an amino acid sequence identical to amino acids 1458 to 1683 of SEQ ID NO: 2.

34. A method for inducing immune tolerance to FVIII in a subject, comprising administering to the subject a composition comprising a chimeric polypeptide, wherein the subject is a fetus, wherein the chimeric polypeptide is suitable for administration to the mother of the fetus, and wherein the polypeptide comprises a FVIII portion and an Fc portion, wherein the FVIII portion consists essentially of or consists of the A2 domain of FVIII, the C2 domain of FVIII, or the A2 domain and the C2 domain of FVIII.

35. The method of claim 34, wherein the Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

36. The method of claim 34 or 35, wherein the Fc portion comprises an amino acid sequence identical to amino acids 1458 to 1683 of SEQ ID NO: 2.

37. A kit when used for reducing an inhibitory immune response to FVIII, said kit comprising: (a) a pharmaceutical composition comprising a chimeric polypeptide which comprises a FVIII portion and a first Fc portion and a pharmaceutically acceptable carrier, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1457 of SEQ ID NO: 2 and wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2, and (b) instructions to administer the composition to a subject in need of reducing an inhibitory immune response to FVIII.

38. The kit of claim 37, wherein the chimeric polypeptide is a FVIII monomer dimer hybrid.

39. The kit of claim 37 or 38, wherein the instructions further include at least one step to identify a subject in need of reducing an inhibitory immune response to FVIII.

40. The kit of claim 39, wherein the at least one step to identify the subject in need of reducing an inhibitory immune response to FVIII includes one or more steps selected from the group consisting of: (a) identifying a subject having a mutation or deletion in the FVIII gene; (b) identifying a subject having a rearrangement in the FVIII gene; (c) identifying a subject having a relative who has previously developed an inhibitory immune response against FVIII; (d) identifying a subject receiving interferon therapy; (e) identifying a subject receiving anti-viral therapy; (f) identifying a subject having a genetic mutation in a gene other than the gene encoding FVIII which is linked with an increased risk of developing an inhibitory immune response; and (g) any combination thereof.

41. The kit of claim 40, wherein the genetic mutation in a gene other than the gene encoding FVIII comprises one or more mutations selected from the group consisting of: (a) a genetic polymorphism associated with increased TNF-aI; (b) a genetic polymorphism associated with increased IL10; (c) a genetic polymorphism associated with decreased CTLA-4; (d) a mutation in DR15 or DQB0602 MHC Class II molecules; and (e) any combination thereof.

42. Use of a chimeric polypeptide comprising a FVIII portion and a first Fc portion, in the manufacture of a medicament for reducing an inhibitory Factor VIII (FVIII) immune response in a subject, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1475 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

43. Use of a chimeric polypeptide comprising a FVIII portion and a first Fc portion, in the manufacture of a medicament for preventing or inhibiting development of an inhibitor to FVIII in a subject, wherein the FVIII portion comprises an amino acid sequence at least 95% identical to amino acids 20 to 1457 of SEQ ID NO: 2, and wherein the first Fc portion comprises an amino acid sequence at least 95% identical to amino acids 1458 to 1684 of SEQ ID NO: 2.

44. Use of a chimeric polypeptide in the manufacture of a medicament for inducing immune tolerance to FVIII in a subject, wherein the subject is a fetus, wherein the medicament is suitable for administration to the mother of the fetus, and wherein the polypeptide comprises a FVIII portion and an Fc portion, wherein the FVIII portion consists essentially of or consists of the A2 domain of FVIII, the C2 domain of FVIII, or the A2 domain and the C2 domain of FVIII.